Anti arnt2

For immunoprecipitations, cells were washed twice in PBS and lysed in 20 mM HEPES, pH 8.0, 420 mM NaCl, 0.5% Igepal, 25% glycerol, 0.2 mM EDTA, 1.5 mM MgCl2, 1 mM DTT and protease inhibitors (Sigma). 100 µg of protein whole cell extract was diluted to 1 mg/ml in immunoprecipitation (IP) buffer (20 mM HEPES, pH 8.0, 150 mM NaCl, 150 mM KCl, 0.1% glycerol, 1 mM EDTA, 1 mM DTT and protease inhibitors) and incubated with 50 µL of BSA blocked Flag M2 resin (Sigma). The resin was washed twice with 1 mL of IP wash buffer (250 mM NaCl, 20 mM HEPES pH 8.0, 0.1% Igepal, and 1 mM EDTA) and the bound material boiled in 20 µl of SDS sample buffer. Input sample (10%) and immunoprecipitations were then run on 7.5%SDS-PAGE gels and transferred to nitrocellulose. Lysates from reporter gene assays were separated on 7.5% SDS-PAGE gel and transferred to nitrocellulose. Proteins were detected using the anti-ARNT2 (Santa Cruz), anti-FLAG (Sigma), anti-Myc (4A6, Upstate) and anti-α-Tubulin antibodies (MCA78G, Serotec). Primary antibodies were detected using horseradish peroxidise-conjugated secondary antibodies and visualised using chemiluminescence. Quantification of ARNT2 co-immunoprecipition band intensity was estimated using ImageLab software (BioRad).

Cells were harvested, washed with PBS and cell lysis was performed in 50 mM Tris–HCl pH 7.4 buffer containing 1% Triton X-100, 150 mM NaCl, 0.5 mM EGTA, 0,5 mM EDTA and anti-protease cocktail (Complete Protease inhibitor Cocktail Tablets, Roche, France). Protein extracts (30 μg) were separated by SDS-PAGE and transferred to Hybond-C Extra nitrocellulose membranes (GE Healthcare, USA) as described [70 (link)]. The following antibodies were used for immunoblotting: anti-actin (Millipore Chemicon, 1:10000), anti-ARNT2 (Santa Cruz, 1:2000), anti-histone H3 (Abcam, 1:50000), anti-trimethyl-histone H3 (Lys 4) (Cosmobio, 1:500), and anti-trimethyl-histone H3 (Lys 27) (Upstate-Millipore, 1:3000). The secondary antibodies were anti-mouse IgG (Santa Cruz Biotechnology, 1:10000) and anti-rabbit IgG (GE Healthcare, 1:10000). Signal detection was performed with the ECL + chemiluminescence detection system (PerkinElmer, France). Densitometric analysis was achieved using ImageJ software.

Lysates were prepared in 20mM HEPES pH 8.0, 420mM NaCl, 0.5% Igepal, 25% Glycerol, 0.2mM EDTA, 1.5mM MgCl₂, 1mM DTT, and protease inhibitors, separated on 7.5–10% SDS-PAGE gel and transferred to nitrocellulose. Proteins were detected using anti-FLAG (Sigma, M2), anti-ARNT1 (Abcam, ab2), anti-P53 (Calbiochem, Ab-1) anti-ARNT2 (Santa Cruz, M-165) and anti-α-Tubulin antibodies (Serotec, MCA78G). Primary antibodies were detected using horseradish peroxidise-conjugated secondary antibodies and visualised using chemiluminescence.