Sigirr

Whole cell lysates were prepared in a lysis buffer containing Tris-HCl (25 mM, pH 7.4), sodium dodecyl sulfate (SDS, 0.1%), Igepal (1%), ethylene diamine tetra acetic acid (2 mM), phenyl methyl sulfonyl fluoride (1 mM), sodium orthovanadate (0.2 mM), NaF (50 mM), NaCl (150 mM), leupeptin (1 μg/ml), and pepstatin (1 μg/ml). Approximately 25 μg of protein was fractionated on SDS-PAGE gels and the separated proteins were electro-transferred onto nitrocellulose membranes. After blocking non-specific sites with 5% skimmed milk in 0.4% Tween-20 in Tris-buffered saline (TBST), the membranes were incubated overnight at 4 °C with primary antibodies in TBST. The dilutions of primary antibodies against Phospho-SP1 (Abcam), SP1 (Abcam), SIGIRR (Santa Cruz Biotech) and mouse ST2 (Abcam) were made as recommended by the manufacturers. Afterwards, the membranes were washed and incubated with HRP-conjugated-anti-rabbit-IgG antibody (1:5,000 in TBST) for 2 h at room temperature. Protein bands were identified by SuperSignal West Femto detection reagent (Thermo Fisher Scientific, Rockford, IL). The membranes were also re-probed with anti-actin antibody (Sigma; 1:5,000 in TBST) after stripping them in a solution containing SDS (10%), Tris (0.5 M), and β-mercaptoethanol (35 μl/ml) at 60 °C for 45 min. The densitometric readings for immunoreactive protein bands were normalized with those for actin.