Anti clock

Total cell lysates of SRA-hLECs were prepared in ice-cold radioimmune precipitation buffer (RIPA buffer), and protein blot analysis was performed as described previously [37 (link),40 (link),41 (link),59 (link),60 (link)]. The membranes were probed with anti-Bmal1 (sc-365645, Santa Cruz Biotechnology, Dallas, TX, USA); anti-Clock (#5157S, Cell Signaling Technology, Danvers, MA, USA); Anti-Nrf2 (sc-365949 Santa Cruz Biotechnology); Anti-Nrf2 (ab62352, Abcam^®, Cambridge, MA, USA); Anti-Prdx6 antibody (LF-PA0011, Ab Frontier, South Korea), Anti-NQO1 (ab28947, Abcam^®, Cambridge, MA, USA), Anti-HO1 (Ab13248, Abcam^®, Cambridge, MA, USA), Anti-SOD1 (sc-515404, Santa Cruz Biotechnology, Dallas, TX, USA), Anti-SOD2 (sc-137254, Santa Cruz Biotechnology, Dallas, TX, USA), or β-actin (A2066, Sigma-Aldrich, St. Louis, MO, USA)/Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH)(sc-365062, Santa Cruz Biotechnology, Dallas, TX, USA)/Tubulin (Abcam, Cambridge, MA, USA) as an internal control to monitor those protein expressions. After secondary antibody (sc-2354 and sc-2768, Santa Cruz Biotechnology, Dallas, TX, USA), protein bands were visualized by incubating the membrane with luminol reagent (sc-2048; Santa Cruz Biotechnology, Dallas, TX, USA), and images were recorded with a FUJIFILM-LAS-4000 luminescent image analyzer (FUJIFILM Medical Systems Inc., Hanover Park, IL, USA).

Cells were immediately placed on ice and washed with ice-cold PBS. All wash buffers and the final resuspension buffer included 1x protease inhibitor mixture (GE Healthcare), NaCl (150 μM), β-glycerophosphate (62.5 mM), DTT (0.1 μM), NaF (5 mM), and Na₃VO₄ (200 μM). When needed, CelLytic NuCLEAR extraction kit (Sigma Aldrich) was used to generate nuclear proteins. Immunoprecipitation was performed using Protein A/G PLUS-Agarose beads (SantaCruz) following standard protocol. Proteins were resolved on 8–12% SDS-polyacrylamide gels and transferred by electroblotting to PVDF membranes (Bio-Rad). The membranes were blocked with 5% nonfat dry milk in TBST (50 mM Tris pH 7.6 with 0.1% Tween 20) and incubated overnight at 4°C in 5% nonfatdry milk in TBST with antibody. Immunolabeling was detected using the ECL reagent (Amersham Biosciences). Relative expression levels were determined by quantitative densitometric analysis using one-dimensional image analysis software (GE Healthsciences). Antibodies used were anti-BMAL1 (#14020, Cell Signaling), anti-CLOCK (#5157, Cell Signaling; #3517, Abcam), anti-RORα (#NBP1-52813, Novus), anti-HIF-1α (#MAB1536, R&D Systems; #ab2185, Abcam), anti-HIF-1β/ARNT (#5537, Cell Signaling), anti-GAPDH (#NB300-221SS, Novus) and anti-Lamin A/C (#2032, Cell Signaling).