Anti bmal1

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).

Total‐cell protein was extracted using RIPA buffer with protease inhibitors and phosphatase inhibitor (Life Technologies). The BCA Protein Assay Kit (Beyotime Bio‐ technology) was used to quantify the protein concentration. An equal amount of protein was loaded onto 8%–12% SDS‐PAGE and then transferred to PVDF membrance (Hercules bio rad). After incubation with 5% skimmed milk at room temperature for 1 h, the membrance was incubated with the following antibodies: anti‐BMAL1, anti‐E‐cadherin, anti‐N‐cadherin, anti‐vimentin, anti‐β‐catenin, anti‐phospho‐MEK, anti‐phospho‐ERK1/2 (Cell Signaling Technology), anti‐p38, anti‐JNK, anti‐c‐Myc, anti‐RAF, and anti‐Ki67 (Abcam). Anti‐GAPDH mouse monoclonal antibody (Cell Signaling Technology) was used as load control. The ECL system was used for blots (Amersham Biosciences).

Immunoblot analysis for U2OS cells was performed as described (61 (link)) using the following antibodies: anti-BMAL1 (14020, Cell Signaling, Danvers, MA, USA), anti-CLOCK (sc-6927, Santa Cruz Biotechnology, Dallas, TX, USA), anti-PER2 (20359-1-AP, Proteintech, Chicago, IL, USA), CRY1 (A302-614A, Bethyl Laboratories, Montgomery, TX, USA), anti-CRY2 (13997-1-AP, Proteintech), anti–REV-ERBα (13418, Cell Signaling), anti–REV-ERBβ (GTX115322, GeneTex, Irvine, CA, USA), anti-CCNB1 (#4138, Cell Signaling), anti-CCND1 (ab134175, Abcam, Cambridge, MA, USA), anti-CDK4 (12790, Cell Signaling), anti-AKT (4685S, Cell Signaling), anti-phospho AKT Ser⁴⁷³ (pAKT) (4060S, Cell Signaling), anti-p44/42 mitogen-activated protein kinase (MAPK) (Erk1/2) (4695S, Cell Signaling), anti–phospho-p44/42 MAPK (Erk1/2)-Thr²⁰²/Tyr²⁰⁴ (pERK) (4370S, Cell Signaling), anti–caspase-3 (9662S, Cell Signaling), anti-HSF1 (sc-17756, Santa Cruz Biotechnology), anti-HSP70 (4872S, Cell Signaling), anti-HSP90AA1 (8165S, Cell Signaling), anti-HSP90AB (7411S, Cell Signaling), anti-HSP90B1 (Grp94) (2104S, Cell Signaling), and anti-TRAP1 antibody (GTX102017, GeneTex). Anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (sc25778, Santa Cruz Biotechnology), anti-tubulin (ab18251, Abcam), and anti–β-actin (4967S, Cell Signaling) were used as loading control antibodies.

Mouse lung tissues were lysed in protein sample buffer (50 mM Tris-Hcl, 10% glycerol, 2% SDS, 100 mM DTT, 5% β-mercaptoethanol and bromophenol blue to colour, pH 6.8) with cOmplete EDTA-free protease inhibitor cocktail (Merck). Protein samples were quantified using Bradford protein assay, and then electrophoresed on 4-20% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad). Subsequently, gels were transferred to nitrocellulose membranes, then blocking step was conducted in Odyssey Blocking Buffer (LI-COR) for one hour, followed by incubation with primary antibodies (either anti-BMAL1, Cell Signaling; or monoclonal anti-α-Tubulin, Merck) overnight at 4°C. Secondary antibodies IRDye 680/800 were used and protein signals were analysed using LI-COR Odyssey CLx.

Small molecules that were elected for further oscillation tests were purchased from the following companies: ISX-9 from Selleck; GW4064, OSI-930, Atrasentan, Forskolin, and KN93 from Med Chem Express; 2-NP and HhAntag from TargetMol. Antibodies were purchased from the following resources: anti-BMAL1 (14020, Cell Signaling Technology), anti-DBP (12662-1-AP, ProteinTech), anti-PER2 (13168, ABclonal), anti-REV-ERBα (13418, Cell Signaling Technology), anti-GAPDH (60004, ProteinTech), HRP-conjugated goat anti-rabbit secondary antibody (1706515, Bio-Rad), and HRP-conjugated goat anti-mouse secondary antibody (1706516, Bio-Rad).