Immunoblot Analysis of Signaling Pathways

Cells were lysed in RIPA buffer with added protease inhibitors and protein content was measured by BCA assay. Total protein was resolved using SDS-PAGE and subjected to immunoblot analysis. Nuclear and cytoplasmic protein fractions were separately processed similar to total protein and analyzed by immunoblot. Densitometric analyses were performed using ImageJ and phospho-p65 values were normalized to total p65. Uncropped blots are included with supplemental data. Antibodies used were phospho-STAT3 (1:1000, Cell Signaling #9131S), phospho-STAT5 (1:1000, Cell Signaling 9314S), phospho-p65 (1:1000, Cell Signaling #3033S), total STAT3 (1:1000, Cell Signaling #12640), total STAT5 (1:1000, Cell Signaling #94205), total p65 (1:1000, Cell Signaling #8242), GAPDH (1:10,000, Cell Signaling #2118), β-tubulin (1:1000, Cell Signaling #2146S), Lamin B1 (1:1000, Cell Signaling #12586S). We detected two bands for total STAT5 proteins, which correspond to Stat5a and STAT5b, isoforms of STAT5 encoded by homologous genes^{24 (link)}. We also detected two bands for p65, which may correspond to one of the reported isoforms for p65, of which the larger one is transcriptionally active^{25 (link)}.