Protein isolation, immunoprecipitation, and western blotting

Cells were homogenized in ice-cold immunoprecipitation lysis buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA) supplemented with inhibitors of phosphatase activity (1 mM PMSF, 1 mM Na3VO4, 1 mM NaF), and a protease inhibitor cocktail (Sigma, St. Louis, MO, USA). The protein concentration was determined using a BCA assay with BSA as a standard. All samples were denatured and reduced in sample buffer (0.555 mM bis-Tris, 4.44% sodium dodecyl sulfate, 0.333 mM HCl, 30% glycerol, 2.22 mM EDTA, 10% β-mercapto-ethanol, 0.04% bromophenol blue) and heated for 10 min at 90°C. Proteins were separated on a 12% bis-Tris-PAGE gel run for 1.5 h at 120 V. Samples were transferred to nitrocellulose membranes for 2.5 h at 150 mA followed by blocking in 5% milk for 2 h. Next, the membranes were incubated overnight at 4 °C with antibodies against SP1 and ACTB (Abcam, Cambridge, MA, USA). The membranes were then washed three times in Tris-buffered saline + 1% Tween 20 followed by a 2-hour incubation at room temperature with a species-specific secondary antibody conjugated to horseradish peroxidase (Jackson Immunoresearch, West Grove, PA, USA). Protein-antibody bound complexes were visualized by chemiluminescence using horseradish peroxidase conjugated secondary antibodies. Detection was performed using a BioRad ChemiDoc XRS System and the signal intensity was analyzed and quantified with BioRad Image Lab Software. Protein expression was normalized to the expression of ACTB (loading control), for each sample.

Total cell extracts were used to immunoprecipitate SP1 (Abcam) or GATA4 (Cell Signaling, Danvers, MA, USA) using previously described protocols [28]. Western blotting of immunoprecipitated proteins was performed as described above.