Chemiluminescent hrp substrate

Cells were solubilized in RIPA lysis buffer containing protease and phosphatase inhibitors. Protein concentrations were determined using the BCA Protein Quantitation Assay Kit (KeyGEN, China). Proteins were separated on a 10% SDS-polyacrylamide gels and transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were probed with anti-NPAS3 antibody (1:1000, Abcam, Cambridge, MA, USA), anti-VGF antibody (1:1000, Abcam, Cambridge, MA, USA), anti-NF-κB (p65) antibody (1:1000, Cell Signaling, Boston, MA, USA), anti-NF-κB (p52) antibody (1:1000, Cell Signaling, Boston, MA, USA), anti-β-Actin antibody (1:1000, Cell Signaling, Boston, MA, USA), anti- GAPDH antibody (1:2000, ZSGB-Bio, China), anti-PKD (1:500, Cell Signaling, Boston, MA, USA), anti-phospho-PKD (1:250, Cell Signaling, Boston, MA, USA), anti-CaMKII (1:500, Cell Signaling, Boston, MA, USA), anti-phospho- CaMKII (1:250, Cell Signaling, Boston, MA, USA) overnight, respectively. Membranes were washed, followed by incubation with goat anti-rabbit horseradish peroxidase-conjugated IgG (1:5000, abbkine) at room temperature for 1 h. Proteins were detected using Chemiluminescent HRP Substrate (Advansta) and visualized with the ECL detection system (Bio-Rad, Berkeley, CA, USA). The bands were measured by Gel-Pro Analyzer software (Media Cybernetics, Rockville, MD, USA).

Cell lysates were prepared in cell lysis buffer (Sigma) containing 50 mM sodium fluoride, 0.2 mM sodium orthovanadate, and 1% protease inhibitor. Total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Millipore, Bedford, MA, USA). After blocking with 5% skim milk at room temperature for 1 h, the membranes were incubated with primary antibodies against UCP1 (Abcam, Cambridge, MA, USA), PGC-1α (Boster Bio, Pleasanton, CA), TFAM (Cell Signaling, Beverly, MA), NDUFB8 (Invitrogen, Carlsbad, CA, USA), SDHB (Invitrogen), UQCRC2 (Abcam), COXIV (Abcam), ATP5A (Abcam), p38 MAPK (Cell Signaling), phospho-p38 MAPK (Cell Signaling), CREB (Abcam), phospho-CREB (Abcam), AKT (Abcam), phospho-AKT (Abcam), phospho-AMPK (Cell Signaling), and β-actin (Sigma) at 4°C overnight. The membranes were then incubated with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG antibodies at room temperature for 30 min. Membranes were developed with chemiluminescent HRP substrate (Advansta Inc, Menlo Park, CA, USA) using X-ray films.

Cells were lysed using radioimmunoprecipitation assay buffer (RIPA buffer; 50 mM Tris−HCl, 150 mM sodium chloride (NaCl), 1% NP−40, 0.1% sodium dodecyl sulfate (SDS), a protease inhibitor cocktail, 50 mM sodium fluoride, and 0.2 M sodium orthovanadate). After centrifugation (12,000× g for 15 min), the protein concentration of the supernatant was measured. The proteins in the cell lysate were measured using the Braford assay. Next, 20 μg of protein from each sample was electrophoresed on 10% acrylamide SDS−polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Millipore, Burlington, MA, USA). Membranes were then blocked in 5% skimmed milk for 1 h at room temperature and incubated with primary and secondary antibodies. The primary antibodies were anti−UCP1 (Abcam, Waltham, MA, USA), anti−PGC1α (Boster Bio, Pleasanton, CA, USA), anti−adipose triglyceride lipase (ATGL; Boster Bio), anti−phospho hormone−sensitive lipase (HSL; Cell Signaling, MA, USA), anti−TFAM (Invitrogen, Carlsbad, CA, USA), anti−NRF1 (Invitrogen), anti−AMPK (Cell Signaling), anti−phospho−AMPK (Cell Signaling), anti−AKT (Cell Signaling), anti−phospho−AKT, and β−actin (Sigma−Aldrich); proteins were visualized using chemiluminescent HRP substrate (Advansta Inc., San Jose, CA, USA).