LPS group). Picture_2.TIF (670K) GUID:?F4D6E18F-6882-4C64-BADD-D005F5330D20 Supplementary Body 3: Ramifications of licochalcone A on mammary gland in LPS-induced mice mastitis. mammary gland in LPS-induced mice mastitis. Mammary gland tissue from each experimental group (= 10) had been attained at 24 h after LPS administration. Mammary gland tissue of (A) control group, (B) LPS group, (C) LPS + licochalcone A (5 mg/kg) group, (D) LPS + licochalcone A (10 mg/kg) group, and (E) LPS + licochalcone A (15 mg/kg) group. Picture_3.jpg (959K) GUID:?D88BB1A1-50D7-4868-809F-AEDDB6CAFDC3 Abstract Background/Aims: Mastitis can be an severe scientific inflammatory response. The incident and advancement of mastitis significantly disturb women’s physical and mental wellness. Licochalcone A, a phenolic substance in and outcomes demonstrated that licochalcone A reduced the histopathological impairment as well as the inflammatory replies considerably, and improved integrity of blood-milk hurdle. The results confirmed that licochalcone A inhibited LPS-induced inflammatory replies and raise the protein degrees of ZO-1, occludin, and claudin3 in mMECs. The and mechanistic research discovered that the anti-inflammatory aftereffect of licochalcone A in LPS-induced mice mastitis was mediated by MAPK and AKT/NF-B signaling pathways. Conclusions and Implications: Our tests collectively indicate that licochalcone A secured against LPS-induced mice mastitis via enhancing the bloodCmilk hurdle integrity and inhibits the inflammatory response by MAPK and AKT/NF-B signaling pathways. in 1975 by Saitoh (9). The framework of licochalcone A and different biological activities have already been set up to time (Body 1) (10), including anti-inflammatory (11), anti-oxidation (12), antiseptic, and anti-tumor properties (13, 14). As a normal Chinese medication monomer with anti-inflammatory and anti-bacterial properties (15, 16), they have many advantages over traditional antibiotics. Medication resistance to the compound is fairly uncommon (17), plus a significant inhibitory influence on irritation. Therefore, we speculate that licochalcone A may possess a protective influence on mastitis also. Open up in another window Body 1 Framework of licochalcone A. The bloodCmilk hurdle is very important to mammary gland level of resistance to the exterior environment (18). Devastation from the bloodCmilk hurdle aggravates infection and promotes the introduction of irritation (19). Acute mastitis is normally caused by infections of gram-negative bacterias and explosive proliferation (20). could cause severe defense response to mammary gland (22). Nevertheless, LPS also destroys the bloodCmilk hurdle and promotes the incident and advancement of irritation (23). So inside our tests, mouse mastitis model was built using LPS extracted from and mastitis versions. Materials and Strategies Medications and Reagents Licochalcone A (purity 98%) was extracted from Chengdu Pufei De Biotech Co., Ltd, dimethylsulfoxide (DMSO) from Sigma Chemical substance Co. (St. Louis, MO, USA), and fetal bovine serum (FBS) from Clark Bioscience Co. (Richmond, va, USA). Dulbecco’s improved Eagle’s moderate (DMEM) for cell lifestyle was extracted from Invitrogen-Gibco (Grand Isle, NY, USA). Cell Keeping track of Package-8 (CCK8) was obtained from Saint-Bio Co. (Shanghai, China). 3,3,5,5-Tetramethylbenzidine, Resorcinol, Hepes and H2O2 had been purchased from Sigma Chemical substance Co. (St. Louis, MO, USA). Enzyme-linked immunosorbent assay (ELISA) sets for TNF-, IL-1 and IL-6 were extracted from Biolegend Co. (NORTH PARK, CA, USA). The principal antibodies AKT, p-AKT, ERK1/2, p-ERK1/2, JNK1/2, p-JNK1/2, P38, p-P38, IB, p-IB, NF-B-p65, and p-NF-B-p-p65 had been bought from Cell Signaling Technology (CST) (Danvers, MA, USA). ZO-1 was obtained from Proteintech (Chicago, IL, USA). Claudin-3, COX-2 and iNOS had been bought from Abcam (Cambrige, UK), occludin from Thermo Fisher (Waltham, MA, USA) and -tubulin from Bosterbio (CA, USA). The supplementary SR-2211 antibodies found in this scholarly research, including Alexa Fluor 594 or Alexa Fluor 488 conjugate donkey anti-rabbit or anti-mouse IgG (H+L) extremely cross-adsorbed supplementary antibody were bought from Life Technology (Carlsbad, CA, USA) and HRP-conjugated goat anti-mouse and goat anti-rabbit supplementary antibodies were bought from Bosterbio. Cell Lifestyle mMECs bought from American Type Lifestyle Collection (ATCC? CRL-3063?) had been cultured in DMEM containing 10% FBS at 37C within a humidified incubator under 5% CO2. Cell Viability The result of licochalcone A on cell viability was motivated using the CCK8 assay. mMECs had been treated with licochalcone A (1.2, 1.8, 2.4, and 3 g/mL), LPS (1 g/mL) or LPS+.Proteins was collected after 0, 15, 30, 45, and 60 min. (A), IL-1 (B), and TNF- (C), as well as the comparative mRNA level was normalized to -actin mRNA. Beliefs are provided as means SD (= 3) (* 0.05, ** 0.01, *** 0.001, and **** 0.0001 vs. LPS group). Picture_2.TIF (670K) GUID:?F4D6E18F-6882-4C64-BADD-D005F5330D20 Supplementary Figure 3: Ramifications of licochalcone A in mammary gland in LPS-induced mice mastitis. Mammary gland tissue from each experimental group (= 10) had been attained at 24 h after LPS administration. Mammary gland tissue of (A) control group, (B) LPS group, (C) LPS + licochalcone A (5 mg/kg) group, (D) LPS + licochalcone A (10 mg/kg) group, and (E) LPS + licochalcone A (15 mg/kg) group. Picture_3.jpg (959K) GUID:?D88BB1A1-50D7-4868-809F-AEDDB6CAFDC3 Abstract Background/Aims: Mastitis can be an severe scientific inflammatory response. The incident and advancement of mastitis significantly disturb women’s physical and mental wellness. Licochalcone A, a phenolic substance in and outcomes demonstrated that licochalcone A considerably reduced the histopathological impairment as well as the inflammatory replies, and improved integrity of blood-milk hurdle. The results confirmed that licochalcone A inhibited LPS-induced inflammatory replies and raise the protein degrees of ZO-1, occludin, and claudin3 in mMECs. The and mechanistic research discovered that the anti-inflammatory aftereffect of licochalcone A in LPS-induced mice mastitis was mediated by MAPK and AKT/NF-B signaling pathways. Conclusions and Implications: Our tests collectively indicate that licochalcone A secured against LPS-induced mice mastitis via enhancing the bloodCmilk hurdle integrity and inhibits the inflammatory response by MAPK and AKT/NF-B signaling pathways. in 1975 by Saitoh (9). The framework of licochalcone A and different biological activities have already been founded to day (Shape 1) (10), including anti-inflammatory (11), anti-oxidation (12), antiseptic, and anti-tumor properties (13, 14). As a normal Chinese medication monomer with anti-inflammatory and anti-bacterial properties (15, 16), they have many advantages over traditional antibiotics. Medication resistance to the compound is fairly uncommon (17), plus a significant inhibitory influence on swelling. Consequently, we speculate that licochalcone A could also possess a protective influence on mastitis. Open up in another window Shape 1 Framework of licochalcone A. The bloodCmilk hurdle is very important to mammary gland level of resistance to the exterior environment (18). Damage from the bloodCmilk hurdle aggravates infection and promotes the introduction of swelling (19). Acute mastitis is normally caused by disease of gram-negative bacterias and explosive proliferation (20). could cause severe defense response to mammary gland (22). Nevertheless, LPS also destroys the bloodCmilk hurdle and promotes the event and advancement of swelling (23). So inside our tests, mouse mastitis model was built using LPS extracted from and mastitis versions. Materials and Strategies Medicines and Reagents Licochalcone A (purity 98%) was from Chengdu Pufei De Biotech Co., Ltd, dimethylsulfoxide (DMSO) from Sigma Chemical substance Co. (St. Louis, MO, USA), and fetal bovine serum (FBS) from Clark Bioscience Co. (Richmond, va, USA). Dulbecco’s customized Eagle’s moderate (DMEM) for cell tradition was from Invitrogen-Gibco (Grand Isle, NY, USA). Cell Keeping track of Package-8 (CCK8) was obtained from Saint-Bio Co. (Shanghai, China). 3,3,5,5-Tetramethylbenzidine, Resorcinol, H2O2 and Hepes had been bought from Sigma Chemical substance Co. (St. Louis, MO, USA). Enzyme-linked immunosorbent assay (ELISA) products for TNF-, IL-6 and IL-1 had been from Biolegend Co. (NORTH PARK, CA, USA). The principal antibodies AKT, p-AKT, ERK1/2, p-ERK1/2, JNK1/2, p-JNK1/2, P38, p-P38, IB, p-IB, NF-B-p65, and p-NF-B-p-p65 had been bought from Cell Signaling Technology (CST) (Danvers, MA, USA). ZO-1 was obtained from Proteintech (Chicago, IL, USA). Claudin-3, COX-2 and iNOS had been bought from Abcam (Cambrige, UK), occludin from Thermo Fisher (Waltham, MA, USA) and -tubulin from Bosterbio (CA, USA). The supplementary antibodies found in this research, including Alexa Fluor 594 or Alexa Fluor 488 conjugate donkey anti-rabbit or anti-mouse IgG (H+L) extremely cross-adsorbed supplementary antibody were bought from Life Systems (Carlsbad, CA, USA) and HRP-conjugated goat anti-mouse and goat anti-rabbit supplementary antibodies were bought from Bosterbio. Cell Tradition mMECs bought from American Type Tradition Collection (ATCC? CRL-3063?) had been cultured in DMEM containing 10% FBS at 37C inside a humidified incubator under 5% CO2. Cell Viability The result of licochalcone A on cell viability was established using the CCK8 assay. mMECs had been treated with licochalcone A (1.2, 1.8, 2.4, and 3 g/mL), LPS (1 g/mL) or LPS+ licochalcone A (1.2, 1.8, 2.4, and 3 g/mL) for 4 h. Subsequently, 10 L CCK8 was put into each well. After 1 h, absorbance (OD) was assessed at 450 nm on the microplate audience (Bio-Rad, CA, USA). Pet Tests BALB/c mice (8~10 weeks outdated, 25~30 g pounds) were bought from the guts of Experimental Pets of Baiqiuen Medical University of Jilin College or university (Jilin, China). Pets had been housed in accredited, regular lab cages and administered food and water before experimental make use of. All animals treatment.Finally, almost all sections had been washed 3 x with PBS and stained with DAPI for 5 min. mouse macrophage (mMECs). The mRNA degrees of IL-6 (A), IL-1 (B), and TNF- (C), as well as the comparative mRNA level was normalized to -actin mRNA. Ideals are shown as means SD (= 3) (* 0.05, ** 0.01, *** 0.001, and **** 0.0001 vs. LPS group). Picture_2.TIF (670K) GUID:?F4D6E18F-6882-4C64-BADD-D005F5330D20 Supplementary Figure 3: Ramifications of licochalcone A about mammary gland in LPS-induced mice mastitis. Mammary gland cells from each experimental group (= 10) had been acquired at 24 h after LPS administration. Mammary gland cells of (A) control group, (B) LPS group, (C) LPS + licochalcone A (5 mg/kg) group, (D) LPS + licochalcone A (10 mg/kg) group, and (E) LPS + licochalcone A (15 mg/kg) group. Picture_3.jpg (959K) GUID:?D88BB1A1-50D7-4868-809F-AEDDB6CAFDC3 Abstract Background/Aims: Mastitis can be an severe medical inflammatory response. The event and advancement of mastitis significantly disturb women’s physical and mental wellness. Licochalcone A, a phenolic substance in and outcomes demonstrated that licochalcone A considerably reduced the histopathological impairment as well as the inflammatory reactions, and improved integrity of blood-milk hurdle. The results proven that licochalcone A inhibited LPS-induced inflammatory reactions and raise the protein degrees of ZO-1, occludin, and claudin3 in mMECs. The and mechanistic research discovered that the anti-inflammatory aftereffect of licochalcone A in LPS-induced mice mastitis was mediated by MAPK and AKT/NF-B signaling pathways. Conclusions and Implications: Our tests collectively indicate that licochalcone A shielded against LPS-induced mice mastitis via enhancing the bloodCmilk hurdle integrity and inhibits the inflammatory response by MAPK and AKT/NF-B signaling pathways. in 1975 by Saitoh (9). The framework of licochalcone A and different biological activities have already been founded to day (Shape 1) (10), including anti-inflammatory (11), anti-oxidation (12), antiseptic, and anti-tumor properties (13, 14). As a normal Chinese medication monomer with anti-inflammatory and anti-bacterial properties (15, 16), they have many advantages over traditional antibiotics. Medication resistance to the compound is fairly uncommon (17), plus a significant inhibitory influence on swelling. Consequently, we speculate that licochalcone A could also possess a protective influence on mastitis. Open up in another window Amount 1 Framework of licochalcone A. The bloodCmilk hurdle is very important to mammary gland level of resistance to the exterior environment (18). Devastation from the bloodCmilk hurdle aggravates infection and promotes the introduction of irritation (19). Acute mastitis is normally caused by an infection of gram-negative bacterias and explosive proliferation (20). could cause severe defense response to mammary gland (22). Nevertheless, LPS also destroys the bloodCmilk hurdle and promotes the incident and advancement of irritation (23). So inside our tests, mouse mastitis model was built using LPS extracted from and mastitis versions. Materials and Strategies Medications and Reagents Licochalcone A (purity 98%) was extracted from Chengdu Pufei De Biotech Co., Ltd, dimethylsulfoxide (DMSO) from Sigma Chemical substance Co. (St. Louis, MO, USA), and fetal bovine serum (FBS) from Clark Bioscience Co. (Richmond, va, USA). Dulbecco’s improved Eagle’s moderate (DMEM) for cell lifestyle was extracted from Invitrogen-Gibco (Grand Isle, NY, USA). Cell Keeping track of Package-8 (CCK8) was obtained from Saint-Bio Co. (Shanghai, China). 3,3,5,5-Tetramethylbenzidine, Resorcinol, H2O2 and Hepes had been bought from Sigma Chemical substance Co. (St. Louis, MO, USA). Enzyme-linked immunosorbent assay (ELISA) sets for TNF-, IL-6 and IL-1 had been extracted from Biolegend Co. (NORTH PARK, CA, USA). The principal antibodies AKT, p-AKT, ERK1/2, p-ERK1/2, JNK1/2, p-JNK1/2, P38, p-P38, IB, p-IB, NF-B-p65, and p-NF-B-p-p65 had been bought from Cell Signaling Technology (CST) (Danvers, MA, USA). ZO-1 was obtained from Proteintech (Chicago, IL, USA). Claudin-3, COX-2 and iNOS had been bought from Abcam (Cambrige, UK), occludin from Thermo Fisher (Waltham, MA, USA) and -tubulin from Bosterbio (CA, USA). The supplementary antibodies found in this research, including Alexa Fluor 594 or Alexa Fluor 488 conjugate donkey anti-rabbit or anti-mouse IgG (H+L) extremely cross-adsorbed supplementary antibody were bought from Life Technology (Carlsbad, CA, USA) and HRP-conjugated goat anti-mouse and goat SR-2211 anti-rabbit supplementary antibodies were bought from Bosterbio. Cell Lifestyle mMECs bought from American Type Lifestyle Collection (ATCC? CRL-3063?) had been cultured in DMEM containing 10% FBS at 37C within a humidified incubator under 5% CO2. Cell Viability The result.Weighed against control group, mammary gland from LPS group provided serious pathological injuries, such as for example thickening from the alveolar wall structure, inflammatory cell infiltration (Amount 2B). in LPS-induced mice mastitis. Mammary gland tissue from each experimental group (= 10) had been attained at 24 h after LPS administration. Mammary gland tissue of CAB39L (A) control group, (B) LPS group, (C) LPS + licochalcone A (5 mg/kg) group, (D) LPS + licochalcone A (10 mg/kg) SR-2211 group, and (E) LPS + licochalcone A (15 mg/kg) group. Picture_3.jpg (959K) GUID:?D88BB1A1-50D7-4868-809F-AEDDB6CAFDC3 Abstract Background/Aims: Mastitis can be an severe scientific inflammatory response. The incident and advancement of mastitis significantly disturb women’s physical and mental wellness. Licochalcone A, a phenolic substance in and outcomes demonstrated that licochalcone A considerably reduced the histopathological impairment as well as the inflammatory replies, and improved integrity of blood-milk hurdle. The results showed that licochalcone A inhibited LPS-induced inflammatory replies and raise the protein degrees of ZO-1, occludin, and claudin3 in mMECs. The and mechanistic research discovered that the anti-inflammatory aftereffect of licochalcone A in LPS-induced mice mastitis was mediated by MAPK and AKT/NF-B signaling pathways. Conclusions and Implications: Our tests collectively indicate that licochalcone A covered against LPS-induced mice mastitis via enhancing the bloodCmilk hurdle integrity and inhibits the inflammatory response by MAPK and AKT/NF-B signaling pathways. in 1975 by Saitoh (9). The framework of licochalcone A and different biological activities have already been set up to time (Amount 1) (10), including anti-inflammatory (11), anti-oxidation (12), antiseptic, and anti-tumor properties (13, 14). As a normal Chinese medication monomer with anti-inflammatory and anti-bacterial properties (15, 16), they have many advantages over traditional antibiotics. Medication resistance to the compound is fairly uncommon (17), plus a significant inhibitory influence on irritation. As a result, we speculate that licochalcone A could also possess a protective influence on mastitis. Open up in another window Amount 1 Framework of licochalcone A. The bloodCmilk hurdle is very important to mammary gland level of resistance to the exterior environment (18). Devastation from the bloodCmilk hurdle aggravates infection and promotes the introduction of irritation (19). Acute mastitis is normally caused by an infection of gram-negative bacterias and explosive proliferation (20). could cause severe defense response to mammary gland (22). Nevertheless, LPS also destroys the bloodCmilk hurdle and promotes the incident and advancement of irritation (23). So inside our tests, mouse mastitis model was built using LPS extracted from and mastitis versions. Materials and Strategies Medications and Reagents Licochalcone A (purity 98%) was extracted from Chengdu Pufei De Biotech Co., Ltd, dimethylsulfoxide (DMSO) from Sigma Chemical substance Co. (St. Louis, MO, USA), and fetal bovine serum (FBS) from Clark Bioscience Co. (Richmond, va, USA). Dulbecco’s improved Eagle’s moderate (DMEM) for cell lifestyle was extracted from Invitrogen-Gibco (Grand Isle, NY, USA). Cell Keeping track of Package-8 (CCK8) was obtained from Saint-Bio Co. (Shanghai, China). 3,3,5,5-Tetramethylbenzidine, Resorcinol, H2O2 and Hepes had been bought from Sigma Chemical substance Co. (St. Louis, MO, USA). Enzyme-linked immunosorbent assay (ELISA) sets for TNF-, IL-6 and IL-1 had been extracted from Biolegend Co. (NORTH PARK, CA, USA). The principal antibodies AKT, p-AKT, ERK1/2, p-ERK1/2, JNK1/2, p-JNK1/2, P38, p-P38, IB, p-IB, NF-B-p65, and p-NF-B-p-p65 had been bought from Cell Signaling Technology (CST) (Danvers, MA, USA). ZO-1 was acquired from Proteintech (Chicago, IL, USA). Claudin-3, COX-2 and iNOS were purchased from Abcam (Cambrige, UK), occludin from Thermo Fisher (Waltham, MA, USA) and -tubulin from Bosterbio (CA, USA). The secondary antibodies used in this study, including Alexa Fluor 594 or Alexa Fluor 488 conjugate donkey anti-rabbit or anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody were purchased from Life Systems (Carlsbad, CA, USA) and HRP-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies were purchased from Bosterbio. Cell Tradition mMECs purchased from American Type Tradition Collection (ATCC? CRL-3063?) were cultured in DMEM containing 10% FBS at 37C inside a humidified incubator under 5% CO2. Cell Viability The effect of licochalcone A on cell viability was identified using the CCK8 assay. mMECs were treated with licochalcone A (1.2, 1.8,.To clarify the mechanism of licochalcone A about repairing the bloodCmilk barrier integrity, the protein levels tight junction parts (including ZO-1, Occludin, and claudin3) and the localization changes of claudin-3 and occludin were determined by western blot and immunofluorescence assays. as means SD (= 3) (* 0.05, ** 0.01, *** 0.001, and **** 0.0001 vs. LPS group). Image_2.TIF (670K) GUID:?F4D6E18F-6882-4C64-BADD-D005F5330D20 Supplementary Figure 3: Effects of licochalcone A about mammary gland in LPS-induced mice mastitis. Mammary gland cells from each experimental group (= 10) were acquired at 24 h after LPS administration. Mammary gland cells of (A) control group, (B) LPS group, (C) LPS + licochalcone A (5 mg/kg) group, (D) LPS + licochalcone A (10 mg/kg) group, and (E) LPS + licochalcone A (15 mg/kg) group. Image_3.jpg (959K) GUID:?D88BB1A1-50D7-4868-809F-AEDDB6CAFDC3 Abstract Background/Aims: Mastitis is an acute medical inflammatory response. The event and development of mastitis seriously disturb women’s physical and mental health. Licochalcone A, a phenolic compound in and results showed that licochalcone A significantly decreased the histopathological impairment and the inflammatory reactions, and improved integrity of blood-milk barrier. The results shown that licochalcone A inhibited LPS-induced inflammatory reactions and increase the protein levels of ZO-1, occludin, and claudin3 in mMECs. The and mechanistic study found that the anti-inflammatory effect of licochalcone A in LPS-induced mice mastitis was mediated by MAPK and AKT/NF-B signaling pathways. Conclusions and Implications: Our experiments collectively indicate that licochalcone A safeguarded against LPS-induced mice mastitis via improving the bloodCmilk barrier integrity and inhibits the inflammatory response by MAPK and AKT/NF-B signaling pathways. in 1975 by Saitoh (9). The structure of licochalcone A and various biological activities have been founded to day (Number 1) (10), including anti-inflammatory (11), anti-oxidation (12), antiseptic, and anti-tumor properties (13, 14). As a traditional Chinese medicine monomer with anti-inflammatory and anti-bacterial properties (15, 16), it has several advantages over traditional antibiotics. Drug resistance to this compound is relatively uncommon (17), along with a significant inhibitory effect on swelling. Consequently, we speculate that licochalcone A may also have a protective effect on mastitis. Open in a separate window Number 1 Structure of licochalcone A. The bloodCmilk barrier is important for mammary gland resistance to the external environment (18). Damage of the bloodCmilk barrier aggravates bacterial infection and promotes the development of swelling (19). Acute mastitis is usually caused by illness of gram-negative bacteria and explosive proliferation (20). can cause severe immune response to mammary gland (22). However, LPS also destroys the bloodCmilk barrier and promotes the event and development of swelling (23). So in our experiments, mouse mastitis model was constructed using LPS extracted from and mastitis models. Materials and Methods Medicines and Reagents Licochalcone A (purity 98%) was from Chengdu Pufei De Biotech Co., Ltd, dimethylsulfoxide (DMSO) from Sigma Chemical Co. (St. Louis, MO, USA), and fetal bovine serum (FBS) from Clark Bioscience Co. (Richmond, va, USA). Dulbecco’s altered Eagle’s medium (DMEM) for cell tradition was from Invitrogen-Gibco (Grand Island, NY, USA). Cell Counting Kit-8 (CCK8) was acquired from Saint-Bio Co. (Shanghai, China). 3,3,5,5-Tetramethylbenzidine, Resorcinol, H2O2 and Hepes were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Enzyme-linked immunosorbent assay (ELISA) packages for TNF-, IL-6 and IL-1 were from Biolegend Co. (San Diego, CA, USA). The primary antibodies AKT, p-AKT, ERK1/2, p-ERK1/2, JNK1/2, p-JNK1/2, P38, p-P38, IB, p-IB, NF-B-p65, and p-NF-B-p-p65 were purchased from Cell Signaling Technology (CST) (Danvers, MA, USA). ZO-1 was acquired from Proteintech (Chicago, IL, USA). Claudin-3, COX-2 and iNOS were purchased from Abcam (Cambrige, UK), occludin from Thermo Fisher (Waltham, MA, USA) and -tubulin from Bosterbio (CA, USA). The secondary antibodies used in this study, including Alexa Fluor 594 or Alexa Fluor 488 conjugate donkey anti-rabbit SR-2211 or anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody were purchased from.