Ab1392

Cells were resuspended in 100 μL of radio immunoprecipitation assay (RIPA) buffer (Beyotime) supplemented with protease inhibitor cocktail (Roche). Cell lysate was analyzed by using 12% SDS-PAGE and was transferred onto polyvinylidene fluoride membranes. 5B12 [7 (link)] and ab1392 (Abcam) antibodies were applied to detect GP73 and ESE-1 proteins, respectively. Protein level was normalized by GAPDH, a housekeeping protein detected by mouse anti-GAPDH monoclonal antibody (Kang Xiang, Shanghai).

Shanghai R&S Biotechnology Co., Ltd. (Shanghai, China) provided the vascular smooth muscle cells (VSMCs). The VSMCs underwent the culture in DMEM covering 100 U/mL of streptomycin, 100 U/mL of penicillin, and 10% fetal bovine serum (FBS) at 37° C in a 5% CO2 incubating element under humidification. Sigma (St. Louis, MO, USA) provided AngII and anti-bromodeoxyuridine (BrdU). Cell Signaling Technology (Danvers, Massachusetts) offered antibodies for P38 (8690S), P-P38 (4511), ERK (9102S), P-ERK (4695S), JNK (9252S), P-JNK (4668S). Abcam (Cambridge, MA) offered MD2 (ab24182), CD31 (ab119341), α-SMA (ab32575), Collagen III (ab-23445), PCNA (ab29), Vimentin (ab8978), 3-NT (ab191308), NOX4 (ab133303), TNF-α (ab-1392), GAPDH (ab-8245), TRITC-conjugated secondary antibody and PE-conjugated secondary antibody. Santa Cruz (CA, USA) offered SIRT1 (sc-74465). The small molecule MD2 inhibitor, L6H21, was synthesized by our lab with a purity of 98.9% [19 (link)]. L6H21 was dissolved in dimethylsulphoxide for in vitro studies.