Protein a g plus sepharose

Cells were washed once with ice-cold wash buffer (40 mM HEPES [pH 7.4], 150 mM NaCl), and lysed in ice-cold CHAPS buffer (40 mM HEPES [pH 7.4], 150 mM NaCl, 2 mM EDTA, 0.3% CHAPS, phosphatase inhibitor cocktail and protease inhibitor cocktail). The cell debris was removed by centrifugation at 13,000 rpm for 10 min in a microfuge. The soluble fractions of cell lysates were mixed with anti-raptor antibody (4 μg/10 cm dish, Life Technology, Cat. 42–4000, RRID: AB_2533523), and the mixtures were incubated with rotation for 1.5 hr at 4°C. 80 μl of a 50% slurry of protein A/G plus-sepharose (Santa Cruz Biotechnology) was then added and the incubation continued for an additional 1 hr. Immunoprecipitates were washed twice with ice-cold CHAPS buffer, and once with mTOR kinase buffer (25 mM HEPES [pH 7.4], 50 mM KCl, 10 mM MgCl₂). The kinase assays were performed as previously described (Kim et al., 2002 (link)). CaM (2 μM) or/and CaCl₂ (1 mM) were added into the kinase reaction as indicated. CMDZ (8 μM) or Torin 1 (100 nM, Cayman Chemical) was incubated with the reaction mixtures 10 min prior to initiating the reaction by addition of 250 μM ATP (Sigma). The phosphorylation states of S6K or 4EBP1 were detected by immunoblotting.

Anti-AhR antibodies were from Biomol (Plymouth, PA, USA) or Santa Cruz Biotechnology (immunofluorescence, Santa Cruz, CA, USA); anti-β-actin was from Sigma-Aldrich (St. Louis, MO, USA) and anti-Cav-1, anti-Y¹⁴ p-Cav-1 and anti-Gapdh were from Becton-Dickinson (Franklin Lakes, NJ, USA). Protein A/G plus Sepharose was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Small interfering RNAs (siRNA) and scramble sequences for AhR were from Dharmacon (Lafayette, CO, USA). Cholera toxin-β to stain ganglioside GM1 was from Sigma and the anti-transferrin receptor (TfR) antibody was from Novus Biologicals (Littletown, CO, USA). The pcDNA-AhR expression vector was produced and characterized essentially as indicated [52 (link)].