Anti erk

After the breast cancer tissues and cells were harvested, the RIPA lysis buffer (Beyotime Biotechnology) with PMSF was conducted to isolate the total protein. Then a BCA Protein Assay Kit (Beyotime Biotechnology) was carried out to quantify the protein concentrations. A total of 25 μg proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electro-transferred into PVDF membranes (Millipore, Massachusetts, USA). The above membranes were blocked using 5% skimmed milk and then incubated with the primary antibodies, such as anti-TFAP2C (also named AP2 gamma, 1/25000 dilution, Abcam), anti-HER2 (1/1000 dilution, Abcam), anti-Akt (1/500 dilution, Cell Signaling Technology), anti-p-Akt (Ser473, 1/500 dilution, Cell Signaling Technology), anti-ERK (1/1000 dilution, Abcam), anti-p-ERK (ERK1/2, Thr202/Tyr204, 1/1000 dilution, Santa Cruz Biotechnology) and anti-GAPDH (1/1000 dilution, Abcam) overnight at 4 °C. Then the membranes were incubated with a secondary antibody (1/2000 dilution, Abcam) at RT for 1 h. All the bands were visualized using enhanced chemiluminescence (ECL) reagent (ThermoFisher Scientific).

After cells or tissues were lysed, we used a BCA Protein Assay Kit (Boster Bio, USA) to determine the protein concentration. Total protein was separated by SDS-PAGE. We used a constant current to transfer the separated protein to a PVDF membrane. 5% skim milk was applied to block non-specific reaction sites for 1-2 h. Dilute the primary antibody at the concentration recommended by the instructions, immerse the membrane in this liquid, and place at 4° C for at least 8 hours. The primary antibodies: anti-GAPDH (Proteintech, Wuhan, China), anti-p-ERK (CST), anti-ERK (CST), anti-NOX4 (Abcam, Cambridge, UK), anti-p-AMPK (Abcam), anti-AMPK (Abcam). Horseradish peroxidase-conjugated secondary antibodies were diluted at the concentrations recommended by the instructions, and the membranes were incubated for 2h at room temperature. Enhanced chemiluminescence reagent was used to visualize the protein bands in an imaging densitometer (GE, USA).

The reagent RIPA Lysis Buffer (Thermo Fisher Scientific) was used to extract the total protein of the cultivated chondrocyte or isolated cartilage tissue. RIPA was supplemented with protease inhibitor PMSF. Protein concentration was determined by a BCA kit (Thermo Fisher Scientific). Protein was mixed with protein loading buffer (Thermo Fisher Scientific) and loaded to a 12% SDS-PAGE gel for electrophoresis. Afterward, the gel was transferred to PVDF membrane (Thermo Fisher Scientific). After a blocking step with a blocking buffer, the membrane was incubated with primary monoclonal antibodies at 4 °C overnight. After washing, the membrane was incubated with secondary antibody. Finally, signal was detected by an ECL method. The primary antibodies used in this study includes anti-Aggrecan (1:1000, Abcam), anti-Collagen II (1:1000, Abcam), anti-β-actin (1:3000, Abcam), anti-ADAM8 (1:1000, Abcam), anti-p-ERK (1:1000, Abcam), anti-ERK (1:2000, Abcam), anti-p- NF-κB p65 (1:1000, Abcam), anti-NF-κB p65 (1:2000, Abcam), anti-MMP9 (1:1000, Abcam), anti-Notch1 (1:1000, Abcam), anti-Hes1 (1:2000, Abcam). All the primary antibodies were incubated for 4 h at 37 °C. The HRP-conjugated secondary antibody (1:5000, Sigma-Aldrich) was incubated for 2 h at RT.

Total protein was isolated from cell lysates or tissues by using RIPA buffer. The concentration of protein was detected with a BCA protein kit (Thermo Fisher Scientific). Then, proteins (40 μg per lane) were separated with 10% SDS-PAGE gel and then transferred into polyvinylidene fluoride (PVDF, Thermo Fisher Scientific) membranes. After blocking with 3% skim milk for 1 h, the membranes were incubated with primary antibodies at 4°C overnight. Subsequently, membranes were incubated with secondary anti-rabbit antibody (Abcam; 1:5000) at room temperature for 1 h. Membranes were scanned by using an Odyssey Imaging System and analyzed with Odyssey v2.0 software (LICOR Biosciences, Lincoln, NE, United States). The primary antibodies used in this study as follows: anti-E-cadherin (Abcam, Cambridge, MA, United States; 1:1000), anti-vimentin (Abcam; 1:1000), anti-α-SMA (Abcam; 1:1000), anti-smad2 (Abcam; 1:1000), anti-smad3 (Abcam; 1:1000), anti-USP4 (Abcam; 1:1000), anti-cleaved caspase 3 (Abcam; 1:1000), anti-Akt (Abcam; 1:1000), anti-ERK (Abcam; 1:1000) and anti-β-actin (Abcam; 1:1000). β-actin was used as an internal control.

Details of Western blotting were previously described^{18 (link)}. Cells at 80–90% confluence were lysed on ice in radioimmunoprecipitation assay buffer (RIPA; keygen biotech, China) containing PMFS complete protease inhibitor cocktail (keygen biotech, China). Protein concentration was determined by the BCA assay (keygen biotech, China). Equal protein samples (10 μg) were separated on 12% sodium dodecyl sulfate (SDS)/polyacrylamide gels, and transferred onto 0.45 µm polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore, Bedford, MA, USA). The antibodies used were as follow: anti-IMPA2 rabbit monoclonal antibody (1:1000; GeneCopoeis, USA); anti-ERK (1:10,000, Abcam, UK), anti-p38α (1:500, BBI China), anti-JNK1/2/3 (1:500, BBI China), p-ERK (1:500, Cell Signaling, USA), p-p38α (p-Thr180/Tyr182, 1:500, BBI China), and p-JNK1/2/3 (p-Th183/Ty185, 1:500, BBI China). Horseradish peroxidase (HRP)-conjugated goat anti‑rabbit immunoglobulin G (1:1000; BBI China) was used as second antibody and anti-GAPDH mouse monoclonal antibody (1:5000; BBI China) as a loading control. The final protein expression was detected by enhanced chemiluminescence (Bio-rad, Berkeley, CA, USA) according to the manufacturer’s suggested protocols. The band quantification was conducted using ImageJ (National Institutes of Health, Bethesda, MA, USA).