Total stat3

The protein levels of p-STAT3(s727) (cat: #9134, 100kDa, Cell Signaling Technology, Beverly, MA, USA), total STAT3 (cat: #9189, 100 kDa, Cell Signaling Technology) and GAPDH (#ab181602; Abcam) were measured using a Western blot system (SNAP i.d. Protein Detection System; Merck Millipore, Danvers, MD, USA). Splenocytes were harvested from BALB/c mice and then stimulated with VnP-16 (100 μg/mL) or vehicle for 2 hours, and then with IL-6 (10 ng/mL) for 1 hour. Then, whole-cell lysates were prepared. The protein concentration was measured using the BCA assay method (#23235, Thermo), and samples were separated on a 4–12% sodium dodecyl sulfate polyacrylamide gel and transferred to a nitrocellulose membrane (Amersham Pharmacia, Uppsala, Sweden). The primary antibodies p-STAT3 s727 (cat: #9134, 100kDa; Cell Signaling Technology), total STAT3 (cat: #9189, 100 kDa; Cell Signaling Technology), and GAPDH (cat: ab181602, 36kDa; Abcam) were diluted in 0.1% skim milk in Tris-buffered saline Tween-20 and incubated for 20 min at room temperature. The membrane was washed and incubated with horseradish peroxidase-conjugated secondary antibody for 20 min at room temperature. Band density was estimated by image capture densitometry.

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)}.