Nonfat milk

Total protein was extracted by 1000 mL of RIPA buffer from 100 mg tissue. For cells, VSMCs were washed 3 times in PBS and 100 μL RIPA buffer (Cell Signaling Technology, MA, USA) per well (6-well plate) applied to cells. Cell lifter (Corning Costar) was used to mix and lyse the cells, which were subsequently transferred to eppendorf tubes. All of the samples were standardized to 1.0 mg/mL. A total of 20 mL was loaded on a 6% to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis plate and transferred onto a polyvinylidenedifluoride membrane, so the total amount of each sample of one western blot gel was 20 μg. Membranes were blocked with 5% nonfat milk (Becton Dickinson, Franklin Lakes, NJ), washed, and probed with the following primary antibodies: rat anti-human Talin (1:1000; Abcam, Cambridge, United Kingdom), and GAPDH (1:500; Abcam). After washing, membranes were incubated with a horseradish peroxidase-conjugated goat anti-rat (1:2000; Sigma) for 1 h. Then, membranes were washed and developed using an enhanced chemiluminescence kit according to the manufacturer’s instructions (Thermo Fisher). Western blot assays were repeated three times. After scanning the blots using a flatbed scanner, the band intensities were analyzed using the Image-ProPlus software version 6.0 (Media Cybernetics, Inc., Rockville, MD).

Transient transfection was performed when the MIN6 cell confluency reached 75%, and protein extraction was carried out after 40 h. Cdc42-pcDNA3.1, Cdc42-siRNAs, miR-29a mimic, miR-29a inhibitor, and miR-29a-NC were transiently transfected into cells. Protein was electrophoresed on SDS-polyacrylamide gel consisting of 5% stacking gel and 12% separating gel (Solarbio). First, 15 μg of protein was added to each slot. After the proteins were separated, they were transferred to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Billerica, MA, USA) under 200 mA for 1 hour and 15 minutes. Next, 5% nonfat milk (Becton, Dickinson, and Company, Franklin Lakes, NJ, USA) was used for 2 h blocking. Then, the PVDF membrane was incubated overnight at 4°C with diluted (1 : 1000) primary antibodies (Cdc42 antibody, Abcam, Cambridge, MA, USA; β-catenin, Affinity Biologicals, Shanghai, China; and β-actin, Zhongshanjinqiao Company, Beijing, China). Subsequently, the membrane was incubated for 1.5 h at room temperature with diluted (1 : 5000) secondary antibodies (HPR-labeled anti-rabbit IgG of goat, HPR-labeled anti-rat IgG of goat; both were purchased from Zhongshanjinqiao Company). Lastly, the proteins were detected using an EasySee Western Blot Kit (TransGen Biotech, Beijing, China) with a Gel Imaging System (Bio-Rad, Hercules, CA, USA).

Protein isolation with lysis buffer was first executed, followed by electrophoretically separation via SDS-PAGE and then transferring onto PVDF membranes (Millipore, Billerica, MA). Subsequently, the membranes were blocked in 5% non-fat milk (Becton–Dickinson and Company, Suzhou, China) and bred overnight with primary antibodies at 4℃. Then, the membranes were co-incubated for 1 h with secondary antibody at room temperature. ECL chemiluminescent detection system (Thermo Fisher Scientific, Rochester, NY) were thereafter utilized for protein band visualization.

For immunoblotting, A375 cells were seeded at a density of 8 × 10⁵ cells in 6 cm culture dishes, allowed to adhere overnight and treated with 3u compound for 6 h. Then, the cells were washed once with PBS, pelleted and lysed in the lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS), and an EDTA-free protease inhibitor cocktail tablet (Roche, Mannheim, Germany) was added. The protein concentration was determined using an Enhanced BCA Protein Assay Kit (Beyotime Biotechnology). Equal quantities of cell lysate protein (20 ug) were separated by 12% SDS polyacrylamide gel electrophoresis and blotted onto PVDF membranes (Millipore Corp., Billerica, MA, USA). Membranes were blocked with 10% nonfat milk (Becton Dickson, NJ, USA) in Tris-buffered saline-Tween-20 buffer (10 mM Tris, 100 mM NaCl, 0.1% Tween-20) for 1 h at room temperature. The blots were probed overnight at 4 °C with a primary antibody followed by horseradish-peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (Thermo Fishier Scientific) for 1.5 h at room temperature. The epitope was detected with Immobilon Western Chemiluminescent HRP Substrate from Millipore (Whitehouse Station, NJ, USA). Chemiluminescent signals were detected and analyzed using a Tanon-5500 chemiluminescent imaging system (Tanon, Shanghai, China).