Sc 101713

Total protein was isolated from cells and tissues using cell lysis buffer and then centrifuged at 12000 rpm for 6 minutes at 4 °C. Protein concentrations in the supernatants were measured using a BCA protein assay kit (Beyotime Corporation, Shanghai, China). Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% BSA for 1 hour at room temperature, the membranes were incubated with primary antibodies against β-tubulin (1:1500 dilution, #2148, Cell Signaling Technology, U.S.A.), iNOS (1:1000 dilution, ab15323, Abcam Corporation, U.S.A.), ghrelin (1:500 dilution, sc-50297, Santa Cruz, U.S.A.), IκBα (1:1000 dilution, ab32518, Abcam, U.S.A.) and p-IκBα (1:1000 dilution, sc-101713, Santa Cruz, U.S.A.) at RT for 1–2 hours, followed by application of appropriate HRP-conjugated secondary antibodies for 1 hour at RT. Immunoreactive bands were imagined with a DNR Bio-Imaging system based on the manufacturer’s instructions. The expression of cytoplasmic protein was normalized to β-Actin or β-tublin using ImageJ software.

Cells were lysed in M-PER (Thermo-Fisher Scientific) supplemented with COMPLETE protease inhibitor mixture (Roche). Total protein was quantified using the BCA Protein Assay Kit (Pierce). Equal amounts of protein were subjected to SDS-PAGE using 7.5% to 15% Tris-Glycine gradient gels, and blotted onto PVDF membranes (Bio-Rad Laboratories, Inc.). After blocking with 5% skimmed milk, membranes were incubated with primary antibodies against HIF-1α (1:1000, NB100–134, Novus Biologicals), IκBα (1:200; sc-371, Santa Cruz Biotechnology), p- IκBα^S32/36 (1:200; sc-101713, Santa Cruz Biotechnology) or Actin (1:1000, AC-74, Sigma-Aldrich) in Can Get Signal solution (Toyobo). The membranes were then incubated with HRP-conjugated secondary antibody (Promega), and immunoreactive bands were visualized with ECL plus (GE Healthcare) according to the manufacturer’s instructions. Original images of the immunoblots were shown in Supplementary Fig. S4. For quantification of bands, densitometry analysis was performed using ImageJ software (National Institutes of Health).

A total of 40 µg protein extract was boiled for 10 min in SDS sample buffer, separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane by electroblotting. Non-specific reactivity was blocked in non-fat dry milk in Tween-PBS (5% (w/v) milk in phosphate-buffer saline (PBS) (pH 7.4) and 0.005% Tween 20) for 2 h at room temperature. The nitrocellulose membranes were incubated overnight at 4 °C with the following antibodies: (a) anti-FHC (H-53) (1:200; sc-25617, Santa Cruz Biotechnology, Dallas, TX, USA), (b) anti-p65 (C-20) (sc-372, 1:1000; Santa Cruz Biotechnology), (c) anti-HDAC (AV38530, 1:5000; Sigma-Aldrich), (d) anti-HA probe (F-7) (sc-7392, 1:1000; Santa Cruz Biotechnology), (e) anti-γ-Tubulin antibody (C-20) (1:3000; sc-7396, Santa Cruz Biotechnology), (f) anti-phospho-IκBα (Ser 32/36) (1:1000; sc-101713, Santa Cruz Biotechnology), and (g) anti-IκBα (C-21) (1:1000; sc371, Santa Cruz Biotechnology).
Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and immunoreactive bands were visualized with the ECL Western blotting detection system (BioRad, Hercules, CA, USA).