Ab227661

For immunoblotting, proteins were isolated from cells using RIPA buffer. Total protein extracts (15–50 μg) were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membrane. Membranes were then probed with anti-bax (1:2000; #ab32503; Abcam), bcl-2 (1:2000; #ab196495; Abcam), cleaved-caspase-3/caspase-3 (1:2000; #ab184787; Abcam), SM22α (1:2000; #ab14106; Abcam), calponin (1:500; #ab227661; Abcam), SMMHC (1:2000; #ab125884; Abcam), vimentin (1:2000; #ab92547; Abcam), collagen I (1:1000; #ab270993; Abcam), osteopontin (OPN; 1:1000; #ab63856; Abcam), LAMC1 (1:1000; #ab233389; Abcam), EVI1 (1:1000; #SAB2100723; Sigma), AKT3 (1:2000; #ab152157; Abcam), TP53INP1 (1:2000; #ab202026; Abcam), and β-actin (1:2000; #ab8226; Abcam) at room temperature for 1.5 h. Then, membranes were immersed with the HRP-conjugated secondary antibody at room temperature for 1 h. Following this, the BM chemiluminescence blotting system (Thermo Scientific) was used for detection and protein bands were quantified using Image J software (NIH, USA).

VSMCs in each group were collected and lysed in 100 μL of RIPA lysis buffer (Beyotime, Shanghai, China). Then, the total protein was extracted and the protein concentration was determined by Bradford method. The equivalent amount of protein in each group was separated by SDS-PAGE and then transferred onto PVDF membrane (Millipore, Bedford, MA, USA). Then, the membrane was blocked in 5% skim milk at room temperature for 1 h before being incubated with the primary antibody overnight at 4°C. Next, secondary antibody was added and the membranes were incubated at room temperature for 1 h. Electrochemiluminescence substrate A and B solutions (Millipore, Bedford, MA, USA) and Amersham Imager 600 (GE Healthcare, Chicago, IL, USA) were used to visualize the protein bands. The ImageJ software (NIH, Bethesda, Maryland, USA) was utilized for quantifying the protein bands. The antibodies used in this study included anti-PIK3CG (1:1000, 140,307, Abcam, Cambridge, UK), anti-SM-22α (1:1000, ab14106, Abcam, Cambridge, UK), anti-α-SMA (1:1000, ab184705, Abcam, Cambridge, UK), anti-smooth muscle myosin heavy chain 11 (SMMHC) (1:1000, ab133567, Abcam, Cambridge, UK), anti-Calponin (1:1000, ab227661, Abcam, Cambridge, UK), anti-GAPDH (1:3000, 60,004-1-Ig, Proteintech, Wuhan, China), and the secondary antibody (1:2000, SA00001-2, Proteintech, Wuhan, China).