Bardford protein assay

Constructed plasmids encoding pcDNA-myocardin-Flag, pcDNA-SOX9-Myc, pcDNA-SRF were contransfected into HEK293T cell line (ATCC Cat#CRL_3216, RRID: CVCL_0063) or PI3Kγ-KD VSMCs. Cells were lysed 48 h post-transfection, nuclear protein fractions extracted by utilizing a cytoplasmic and nuclear protein extraction kit according to the manufacturer's protocol (Boster Biological Technology, China) were incubated with anti-Myc or anti-Flag or anti-SRF antibody overnight at 4 °C. Normal IgG was acted as a negative control. Protein A + G Agarose beads were added and slowly swung for 2 h at 4 °C.The immunoprecipitates were washed five times with pre-cooling PBS and then mixed with SDS-PAGE buffer, followed by western blotting analysis. For western blotting, whole cell lysates were prepared with lysis buffer containing protease inhibitors and quantified using the Bardford Protein Assay (Bio-Rad). Equal amounts of denatured whole cell lysates of VSMCs were separated by SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes. After blocking, proteins were detected with corresponding primary antibodies and subsequently with appropriate HRP-conjugated secondary antibodies. Immunoreactivity was visualized with enhanced chemiluminescence detection reagents and recorded using ChemiDoc imaging system (Bio-Rad). Protein expression levels were normalized against β-actin.