Ab214362

A549 (1×10⁷) and H358 (1×10⁷) cells were lysed in RIPA buffer (Sigma-Aldrich) followed by homogenization at 4°C for 10 min. Protein concentrations were measured using BCA Protein assay kit (Thermo Fisher Scientific). Proteins (30 μg) were analyzed by 12% SDS-PAGE assays followed by transfer to PVDF membranes. Proteins were incubated with rabbit anti-human ERK1/2 (1:500, ab93125, Abcam, China), pERK (1:1,500, phospho-Thr202/Tyr204, 1:1,000, ab214362, Abcam), TGF-β (1:1,500, ab31013, Abcam), and β-actin (1:1,500, ab8226, Abcam) for 12 h at 4°C. Membranes were incubated with HRP-conjugated goat anti-rabbit IgG (cat. No. 4410; Cell Signaling Technology, USA; 1:2,000) secondary antibodies at 37°C for 2 h. Immunoreactivity was evaluated using ECL western blotting kit (Beyotime Institute of Biotechnology, China). Quantitation of signal intensities was evaluated using UVP EC3 v3.0 software (UVP, LLC, USA).

Proteins of esophageal tissue of ESCC patients, paired adjacent normal tissues, and cultured cells were extracted with RIPA lysis buffer (Thermo Fisher Scientific Inc), supplemented with Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific Inc) and were quantified utilizing BCA Protein Assay Kit (Thermo Scientific). Antibodies of KIF4A (Abcam, ab122227), AKT (CST, 4685S), P-AKT (CST, 4060S), P38 (CST, 8690S), P-P38 (CST, 4511S), ERK1/2 (Abcam, ab17942), P -ERK1/2 (Abcam, ab214362), JNK (Abcam, Ab179461), P-JNK (Abcam, Ab124956), GAPDH (Abcam, ab8245) were incubated overnight at 4 degrees overnight, and then with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology ) for 1 hr at room temperature.

Proteins were extracted from the primary cardiomyocytes in RIPA buffer (1% Triton X-100, 150 mmol/L NaCl, 5 mmol/L EDTA, and 10 mmol/L Tris-HCl, pH 7.0; Solarbio, China) supplemented with a protease inhibitor cocktail (Cat: I3786-1ML, Sigma). The cell lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred electrophoretically to a PVDF membrane (Millipore Corporation, USA). After blocking with 8% milk in PBS, pH 7.5, the membranes were incubated with the following specific primary antibodies of Bax (ab32503), Bcl-2 (ab59348), cytochrome C (ab13575), Smac/Diablo (ab32023), cleaved-capase-3 (ab13847), cleaved-capase-9 (ab2324), p-p38 (ab47363), p38 (ab31828), p-ERK (ab214362), and ERK1/2 (ab196883; all at a dilution of 1:1000, Abcam, UK). After overnight incubation, the appropriate HRP-conjugated anti-rabbit IgG secondary antibody (ab205781, Abcam, all at a dilution of 1:5000) was subsequently applied and immunodetection was achieved using the ECL Plus detection system (Millipore Corporation) according to the manufacturer's instructions. Band intensity was quantified using Image Lab™ Software (Bio-Rad, China). GAPDH (ab8245, Abcam) was used as an internal control.

Proteins were extracted from samples using RIPA Kit (Beyotime Biotechnology, China). The protein concentration was quantified using Bradford method. Proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and thereafter transferred to PVDF membrane which was blocked in Tris‐Buffered Saline Tween (TBST) containing 0.1%Tween‐20 and 5% low‐fat milk for 1 h. The primary antibody was added and incubated overnight at 4°C and then the secondary antibodies were added and incubated at room temperature for 1 h. Lastly, the ECL solution was applied to the membranes, and the membranes together with a film were placed in a visualizer for further analysis. The ImageJ software was used to measure the intensities. Primary antibodies for NRAS (ab154291), BRAF (ab151286), MEK1/2 (ab178886), pMEK1/2 (phospho s218, s222 and s226, ab78132), ERK1/2 (ab17942), pERK1/2 (phospho thr202 and thr204, ab214362), and GAPDH (ab9485) were all purchased from Abcam, whereas antibodies for PI3K‐p110α (#4249), PI3K‐p110β (#3011s), AKT (#9272), pAKT‐ser473 (#4060), pAKT‐thr308 (#2965), PTEN (#4005), Cyclin D1 (#2926), and p27 (#3686) were purchased from Cell Signaling. HRP labeled goat anti‐mouse and goat anti‐rabbit as secondary antibodies were purchased from Beyotime Biotechnology.

Proteins from the human aortic wall tissue of patients, aortic wall of mice, and cultured MOVAs and MAECs were extracted with RIPA lysis buffer (Thermo Fisher Scientific Inc.) supplemented with a protease and phosphatase inhibitor cocktail. Western blotting was performed using antibodies against NAMPT (Abcam, ab236874), P16 (Abcam, ab118459), P21 (Abcam, Ab109520), eNOS (Abcam, ab199956), P-eNOS (Abcam, ab32419), SIRT1 (Abcam, ab110304), MMP-2 (Abcam, ab97779), MMP-9 (Abcam, ab38898), AKT (CST, 4685S), P-AKT (CST, 4060S), P38 (CST, 8690S), P-P38 (CST, 4511S), ERK1/2 (Abcam, ab17942), P-ERK1/2 (Abcam, ab214362), JNK (Abcam, Ab179461), P-JNK (Abcam, Ab124956), and GAPDH (Abcam, ab8245), followed by an incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 hour at room temperature. A gel imaging system (Tanon) and AlphaView software were used to image and analyze the Western blots.