Western bright quantum system

24 h after seeding cells, HNF6-shRNA plasmid (HNF-6 sc-37937-SH, Santa Cruz Biotechnology, USA) was used for knocking-down of HNF6, according to the manufacturer's instructions. Scrambled shRNA plasmid (sc-108060, Santa Cruz Biotechnology) was used as a negative control. Knockdown efficiency was assessed 48 h post-transfection by western blotting as well as by real time PCR. Protein of transfected Hepa-1c1c7 cells was extracted using RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate containing protease and phosphatase inhibitors). Protein samples were subjected to electrophoresis on 10% SDS-polyacrylamide gels and transferred to a nitrocellulose (Amersham Protran Supported 0.45 NC, GE Healthcare). After blocking with 0.5% dry milk in PBS-Tween 0.1%, the membranes were probed with anti-HNF6 (1:500, Santa Cruz Biotechnology, sc-376167), and HSP90 (1:1000, Santa Cruz Biotechnology, sc-13119) antibodies overnight at 4°C. Anti-rabbit and -mouse HRP conjugated antibody was used as a secondary antibody. Detection of the immune complexes was performed using WesternBright Quantum system (Advansta, K-12042, USA) and quantification was done with the Quantity One analysis software (Bio-Rad).

Protein of cultured cells and liver tissue was extracted using RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate containing protease and phosphatase inhibitors). Protein samples (40 μg) were subjected to electrophoresis on 10% SDS-polyacrylamide gels and transferred to a nitrocellulose (Amersham Protran Supported 0.45 NC, GE healthcare). After blocking with 0.5% dry milk in PBS-Tween 0.1%, the membranes were probed with antibodies against GR (1:500, Santa Cruz Biotechnology, sc-1004), HSP90α/β (F-8) (1:1000, Santa Cruz Biotechnology, sc-13119), REV-ERBα (1:200, Santa Cruz Biotechnology, sc-100910), BMAL1 (1:500, Ripperger and Schibler, 2006 (link)), NF-κB p65 (1:200, Santa Cruz Biotechnology, sc-109), IκBα (1:200, Santa Cruz Biotechnology, sc-371), phosphorylated (p-)IκBα (1:200, Santa Cruz Biotechnology, sc-8404), tubulin (1:1000, Abcam, ab 15246) or LAMINB1 (1:500, Santa Cruz Biotechnology, sc-30264) overnight at 4°C. Anti-rabbit-IgG, mouse-IgG and goat-IgG antibody conjugated to horseradish peroxidase (HPR) was used as a secondary antibody. Detection of the immune complexes was performed using Western Bright Quantum system (Advansta) and quantification was done with the Quantity One analysis software (BioRad).

Using RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate containing protease and phosphatase inhibitors), protein of cultured cells and brain tissue was extracted. Proteins were separated on 12.5% SDS-PAGE and transferred to nitrocellulose (Protran BA 83, 0.2 µm pores, GE healthcare). Primary antibodies were incubated over night at 4°C, Anti-rabbit FABP7 1∶250 (Abcam ab27171), Anti-rabbit actin 1∶5000 (Sigma, A5060) and Anti-BMAL1 1∶1000 [42] (link). Detection of the immune complexes was performed using Western Bright Quantum system (Advansta) and quantification was done with the Quantity One analysis software (BioRad). Actin was used for normalization and relative protein levels were calculated by defining maximal protein levels as 1.