Ecl prime western blotting detection reagent

Pre-adipocytes were seeded at a density of 6 × 10⁴ cells per well in 6-well plates. Following differentiation and treatments, cells were washed twice with PBS and incubated on ice with lysis buffer (ELISA lysis buffer, Thermo Fisher Scientific) containing proteinase and phosphatase inhibitor (Cell Signaling technology, Leiden, The Netherlands). Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane (Thermo Fisher Scientific), and incubated with specific primary antibodies. The antibodies used in this study were directed against β-actin (Cell Signaling Technology, 8H10D10, 1:2,000), CBS (Cell Signaling Technology, D8F2P, 1:500), CSE (Abcam, Ab151769, 1:1,000), 3-MST (Abcam, Ab85377, 1:500), ETHE-1 (Abcam, Ab174302, 1:1,000), TST/rhodanese (Abcam, Ab231248, 1:1,000), and PPAR-γ (CST, 2430S, 1:1,000). Signals from HRP-coupled secondary antibodies were detected with ECL Prime Western Blotting Detection Reagent (Sigma-Aldrich), using a luminescent image analyzer (Fusion FX6, Vilber Lourmat, Marne la Vallée, France) and quantified using the ImageJ software (NIH, Bethesda, MD, USA).

To confirm the proteolytic processing of Gag and GagPol polyproteins in HIV virions, Western blot (WB) analysis was performed as previously described [22 (link),24 (link)]. Due to the abnormal sizes of M50I mutant particles (the diameters of the mutant particles were 190~300 nm, while those of Wt were 110~130 nm [22 (link)]) with a defective autoprocessing of Gag and GagPol polyproteins (lacking of the cleaved mature forms of MA (p17), CA (p24), PR, RT, and IN in the virus particles) [22 (link)], we could not find an appropriate internal control for WB. Thus, we used 1 µg of total viral protein to demonstrate impaired autoprocessing as previously described [22 (link),24 (link)]. WB was performed using a rabbit polyclonal anti-Anti-HIV1 p17/p24/Gag antibody (Cat# ab63917, Abcam, Waltham, MA, USA), a mouse monoclonal anti-HIV p24 antibody (Cat# ab9071, Abcam), and a rabbit polyclonal anti-PR antibody (Cat# ab211627 Abcam, Cat# SKU:65-018, As One International, Santa Clara, CA, USA). Protein bands were detected by using the ECL Prime Western Blotting Detection Reagent (MiliporeSigma) with the Azure 300 (Azure Biosystem (Dublin, CA, USA) [22 (link)].

Proteins were isolated from frozen suprarenal aortas using RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing protease inhibitors. Protein extracts were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrically transferred to polyvinylidene difluoride membranes, which were blocked with 10% nonfat dry milk in TBS-0.05% Tween 20 for 1 hour. Then, membranes were incubated overnight at 4°C using below primary antibodies: anti-Phospho-AMPKα (Thr172) antibody (2535, cell signaling technology, 1:1000), anti-AMPKα antibody (2603, cell signaling technology, 1:1000), anti-Phospho-NF-κB P65 (nuclear factor κB P65) (Ser536) antibody (3033, cell signaling technology, 1:1000), anti- NF-κB P65 antibody (4764, cell signaling technology, 1:1000), anti-Phospho -STAT3 (signal transducer and activator of transcription 3) (Tyr705) antibody (Ab76315, 1:20000), anti-STAT3 antibody (Ab119352, 1:1000), anti-β-actin antibody (sc-81178, Santa Cruz Biotechnology, Santa Cruz, Calif, 1:500) and anti-GAPDH antibody (ab-8245, 1:5000). After that, the appropriate secondary antibody was applied. Blots were detected using an ECL Prime Western Blotting Detection reagent (Millipore). The signals were quantified by ImageJ software.