Smad2 overexpression plasmid

Co-immunoprecipitation was performed following the instruction of Pierce Crosslink Immunoprecipitation Kit (Thermo Scientific, 26147). In brief, AoSMCs were transfected with empty vector (VEC) or smad2 overexpression plasmid (Addgene, 11734), or AoSMCs were starved and treated with Nutlin 3a for 24 h. Post transfected or treated AoSMCs were lysed on ice for 30min in IP Lysis/Wash Buffer (included in kit) containing Halt protease inhibitor cocktail (Thermo Fisher Scientific, 87785). Cell lysate were centrifuged at 12000 rpm for 10min at 4 °C. During centrifugation, we performed binding of antibodies to protein A/G plus agarose and crosslinking the bound antibodies following the instruction. After centrifugation, the pre-cleared lysate was added to the antibody-crosslinked resin in the column, and incubated with gentle end-over-end mixing for overnight at 4 °C. The proteins were finally eluted from the column and subjected to SDS-PAGE and Western blot analysis.