Anti c ebpβ

The cells were lysed with RIPA lysis buffer (Millipore-Sigma, Billerica, MA, USA) containing protease inhibitors (Millipore-Sigma). Protein concentration was determined by BCA assays, and 30 µg cell lysates were resolved by SDS-PAGE on 4–12% gradient Bio-Tris gels, transferred to nitrocellulose membranes (Thermo Fisher Scientific), and probed with each of the following antibodies: anti-CLOCK, anti-BMAL1, anti-TIMP3, anti-C/EBP-α, anti-C/EBP-β, anti-TNF-α, anti-NF-κB, anti-phospho-NF-κB, and anti-I-κB (Cell Signaling Technology, Beverly, MA, USA); anti-MMP-1 (courteously provided by Prof. Jin Ho Chung, Seoul National University College of Medicine, Seoul, South Korea); and GAPDH and horseradish peroxidase–conjugated secondary anti-rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA). Western blotting luminol reagent (Santa Cruz Biotechnology) was used to develop the signals. For zymogram analysis, the cultured medium was harvested every 4 or 12 h after synchronization or UV irradiation and loaded on 10% Zymogram (gelatin) Protein Gels (Thermo Fisher Scientific). The gels were incubated at 37°C overnight and stained with 0.5% Coomassie blue (Millipore-Sigma) according to the manufacturer’s instructions.

Harvested cells were washed with phosphate-buffered saline (PBS), and protein was extracted using lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 1% NP-40, 1 mM EDTA, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM Na₃VO₄, 1 mM NaF, 1 mM PMSF and 0.1 mg/ml leupeptin/aprotinin) on ice for 30 min and centrifuged at 15,000g for 30 min. Then, the supernatant was collected for Western blotting. The proteins were separated by sodium dodecyl sulfate-polyacryl amidegel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose blotting membranes (GE Healthcare Life Science, USA). The primary antibodies were incubated with the membrane overnight at 4°C. Horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG secondary antibody (Beyotime, China) was incubated with the membranes for 1 h at room temperature. The proteins on the membranes were detected using a Tanon imaging system (5200; Tanon Science & Technology, China). The primary antibodies used included anti-STAT6, anti-CEBPα, anti-CEBPβ, and anti-β-actin, which were all purchased from Cell Signaling Technology (USA).

Polyclonal anti-Rap1b antibody, polyclonal anti–phospho-Drp1 (Ser637) and (Ser656) antibodies, monoclonal anti–PGC-1α, and anti–C/EBP-β were from Cell Signaling Technology; human/mouse/rat cytochrome c monoclonal antibody was from BD Biosciences; procaspase-3 antibody and procaspase-9 antibody were from Thermo Fisher Scientific; monoclonal anti–cleaved caspase-3 (Asp175), rabbit polyclonal IgG antibodies including anti-mitofusin2 (Mfn2), anti-catalase, anti–manganese superoxide dismutase (Mn-SOD), anti–nuclear respiratory factor-1 (NRF-1), anti–glutathione peroxidase (GSH-Px), and anti-mtTFA were from Santa Cruz Biotechnology; and plasmids containing pcDNA/Rap1b G12V and pcDNA/Rap1b S17N mutant were generated in our laboratory as previously described (10 (link)). Extracellular signal–related kinase 1/2 (ERK1/2) short interfering RNA (siRNA), PGC-1α siRNA, DFC, MitoRed, and MitoSOX were purchased from Invitrogen.