Ripa buffer kit

Quantitative RT-PCR and WB were carried out as previously described [20 (link)]. RT-PCR reagents were purchased from Accurate Biology (Changsha, China), which included the SYBR Green Premix Pro Taq HS qPCR Kit, AG RNAex Pro Reagent, and Evo M-MLV RT Premix Kit. Supplementary Table 1 shows the primer sequences. Beyotime Biotechnology (Shanghai, China) provided WB reagents, such as BeyoECL Plus and RIPA buffer Kit. Proteintech (Wuhan, China) supplied anti-OGT (11576-2-AP) and anti-GAPDH (10494-1-AP) antibodies.

Total proteins from tissues and cells were extracted using a RIPA buffer kit (P0013B; Beyotime) according to the manufacturer's protocols. The procedures of WB were performed according to a previous study.³³ Briefly, the membranes were blocked with 5% bovine serum albumin (BSA) which was dissolved in Tris‐buffered saline with Tween‐20 for 1 h at room temperature. Then the membranes were labeled with primary antibodies, anti‐myosin light chain 2 (MLCK2) antibody (1:1000, #3672; Cell Signaling Technology), anti‐phospho‐myosin light chain 2 (p‐MLCK2) antibody (1:1000, #3674; Cell Signaling Technology), anti‐MYPT1 antibody (1:1000, #2634; Cell Signaling Technology), anti‐p‐MYPT1 (Thr696) antibody (1:1000, PA5‐38297; Invitrogen), anti‐ROCK1 antibody (1:1000, AF0276; Beyotime), anti‐RhoA antibody (1:1000, AF2179; Beyotime), anti‐ROCK2 antibody (1:1000,#47012,Cell Signaling Technology), and GAPDH antibody (1:1000, AF1186; Beyotime), at 4°C overnight, respectively, followed by incubations with corresponding secondary IgG (H + L) (peroxidase/horse radish peroxidase conjugated) antibodies (E‐AB‐1001; Elabscience) at different dilutions for 2 h at room temperature. The protein bands were visualized and captured using a Tanon 1600R imaging system. The optical density of each band was quantified using ImageJ software and normalized to the intensity of GAPDH.