Af6456

Total proteins were extracted from the thoracic aorta tissues using the radioimmunoprecipitation buffer lysis buffer (Roche, Mannheim, Germany). Total protein concentration was measured using the BCA Protein Assay Kit (Pierce, Rockford, USA) according to manufacturer's instructions. The same amount of total proteins were injected into the sample holes of 10% sodium dodecyl sulfonate- (SDS-) polyacrylamide gel electrophoresis (PAGE) and then electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After being blocked with 5% skim milk for 1 h, the membranes were incubated overnight at 4°C with anti-p38 (Affinity Bioscience, USA; AF6456, 1 : 1000), anti-p-p38 (Affinity Bioscience, USA; AF4001, 1 : 1000), anti-JNK (Abcam; ab179461, 1 : 1000), anti-p-JNK, (Abcam; ab124956, 1 : 5000), anti-ERK (Affinity Bioscience, USA; AF0155, 1 : 5000), anti-p-ERK (Affinity Bioscience, USA; AF1015, 1 : 2000), and anti-β-actin (Abcam; ab8226, 1 : 5000). The membranes were then incubated with secondary antibodies, including goat anti-mouse IgG-H&L (HRP) (Abcam; ab205719, 1 : 10000) and goat anti-rabbit IgG-H&L (HRP) (Abcam; ab205718, 1 : 10000). Protein signals were assessed using enhanced chemiluminescence (ECL) western blot detection system (Millipore, Billerica, MA, USA) and densitometric analysis.

Articular cartilage (0.1 g) was weighed and lysed for 30 min on ice with RIPA lysis buffer containing PMSF (R0010). The supernatant was obtained by centrifugation for 20 min at 4 ℃ and 12,000 rpm, that is, the total protein. The protein concentration was measured by a BCA protein assay kit (PC0020, Solarbio). Equal amounts of protein samples were separated by SDS‒PAGE and then transferred to PVDF membranes (IPVH00010, Millipore). The membrane was blocked with 5% skim milk at room temperature for 1 h and then incubated with antibodies against p38MAPK (AF6456, Affinity), p-p38MAPK (AF4001, Affinity), MMP1 (AF0209, Affinity), MMP13 (AF5355, Affinity) and beta actin (AF7018, Affinity) overnight at 4 °C. Then, the membrane was incubated with the secondary antibody at room temperature for 2 h. The protein bands were detected by ECL reagent (WBKLS0100, Millipore) and observed by a Bio-Rad chemiluminescence imaging system (Chemidoc XRS+, Bio-Rad). ImageJ software was used to quantify the scanned image.

After treatment, the uterine tissues (myometrium and endometrium) were lysed with RIPA buffer. After isolation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred to polyvinylidene difluoride (PVDF) membranes. After blocking by 5% defatted milk powder, the membranes were incubated with the primary antibodies against tubulin alpha antibody (AF7010, 1 : 5000, Affinity, USA), ERK1/2 antibody (AF0155, 1 : 1000, Affinity, USA), phospho-ERK1/2 (Thr202/Tyr204) antibody (ab92946, 1 : 500, Affinity, USA), JNK1/2/3 antibody (AF6318, 1 : 500, Affinity, USA), phospho-ERK1/2 (Tyr204) antibody (AF1014, 1 : 500, Affinity, USA), p38 MAPK antibody (AF6456, 1 : 500, Affinity, USA), and phospho-p38 MAPK (Thr180/Tyr182) antibody (AF4001, 1 : 500, Affinity, USA) at 4°C for 24 h. After washing with PBS, the membranes were incubated with the secondary antibodies at room temperature for another 2 h. Lastly, the signals of protein bands were developed with enhanced chemiluminescence (ECL).

ARPE‐19 cells were lysed in a lysis buffer to extract total protein, and the protein concentration was measured by a bicinchoninic acid assay kit (Beyotime, Shanghai, China). Samples were loaded and separated in 10% Bis‐Tris‐polyacrylamide electrophoresis gels. Then, the proteins were transferred to PVDF membranes, which were blocked with 5% nonfat milk followed by incubation with primary antibodies at 4 °C overnight. After incubation with the secondary antibody for 1 h, the Western blot signals were detected by ECL Plus reagents. ImageJ was used to analyze the protein bands. Primary antibodies against Occludin (1:800, Cat No. 17590‐1‐AP), TLR4 (1:800, Cat No. 66350‐1‐Ig), p65 (1:500, Cat No. 10745‐1‐AP), JNK (1:500, Cat No. 24164‐1‐AP), p‐JNK (1:500, Cat No. 80024‐1‐RR) and MYD88 (1:800, Cat No: 23230‐1‐AP) were purchased from Proteintech. Antibodies against p‐p65 (1:1000, AF2006), GADD45G (1:1000, A10286), p‐p38 (1:1000, AF4001), p38 (1:1000, AF6456), GAPDH (1:3000, AF7021), β‐actin (1:3000, AF7018) and OR11H1 (1:1000, DF5186) were purchased from Affinity.