Ahp1117

Antibody against QKI was purchased from Santa Cruz Biotech (sc-103851), USA. Antibodies against QKI5, QKI6, QKI7 were from Millipore (AB9904, AB9906, AB9908), USA. Antibody against Smooth Muscle Myosin Heavy Chain (SMMHC) was from AbD Serotec (Rabbit, AHP1117). Antibodies against SM22 (rabbit, Ab14106) and calponin (rabbit, Ab46794) were from Abcam, UK. Antibodies against GAPDH (mouse) and monoclonal anti-α smooth muscle actin (SMA) (Clone 1A4, A5228) were from Sigma. All secondary antibodies were from Santa Cruz Biotech.

Protein extraction and immunoblotting were performed as described in our previous report [13 (link)]. Briefly, total proteins extracted using RIPA lysis buffer (P0013B, Beyotime, China) and quantified using a BCA Protein Assay Kit (P0012S, Beyotime, China) were boiled for 10 min at 100 °C. Ten to 30 μg of the proteins were separated in 10% SDS-PAGE gel (P0012A, Beyotime, China) and transferred from the gel to a membrane. The membrane was blocked overnight in 5% skimmed milk followed by incubation with primary antibodies against GAPDH (1:2000, 14C10, Cell Signaling Technology, Danvers, MA, USA), SMαA (Rabbit, 1:1000, 14395-1-AP, Proteintech, Rosemont, IL, USA), SM22α (Rabbit, 1:1000, Ab14106, Abcam, UK), SMMHC (Rabbit, 1:1000, AHP1117, AbD Serotec, UK), h1-calponin (Rabbit, 1:1000, Ab78491, Abcam, UK), and OPG (Rabbit, 1:1000, NB100-56505SS, Novus Biologicals, Centennial, CO, USA) overnight at 4 °C. The membrane was washed thrice with 1X PBST, for 10 min per wash, and incubated with the secondary antibody from Santa Cruz Biotechnology (Dallas, TX, USA) for 1 h at room temperature. The membrane was washed an additional 3 times with 1X PBST, the ECL substrate was added, and the target proteins were observed using the compact X4 in the dark room.