For Western blots, cells were pelleted and lysed directly with denaturing gel loading buffer (Tris-HCl 125 mM, pH 6.8, glycerol 20% vol/vol, SDS 4% vol/vol, β-mercaptoethanol 10% vol/vol, and bromophenol blue). The primary and secondary antibodies are described in Table S6 . Western blots were developed by chemiluminescence (Pierce Super Signal West Dura; Thermo Fisher Scientific) and imaged using an Amersham Imager 600 imager (GE Healthcare Life Sciences). Bands were quantified with Gel-Pro Analyzer 4 (Meyer Instruments).
For immunoprecipitations, cells were serum starved for 48 h, and proteins were extracted with lysis buffer (20 mM Hepes, pH 7.5, 50 mM KCl, and 1 mM MgCl2) with 0.5% digitonin and protease inhibitor (cOmplete EDTA-Free; Roche). Insoluble components were removed by centrifugation at 20,000 g. Primary antibodies preadsorbed to protein G-sepharose beads (GE Healthcare) were added to the cell extract, and the mixture was incubated for 2 h at 4°C. After centrifugation, beads were washed with lysis buffer supplemented with 0.1% digitonin before elution in denaturing gel loading buffer for SDS-PAGE electrophoresis and Western blot analysis.
Isolation of mRNA and quantitative mRNA analysis was performed as previously described (Jonassen et al., 2008 (link)) using the primers tabulated inTable S7 .
For immunoprecipitations, cells were serum starved for 48 h, and proteins were extracted with lysis buffer (20 mM Hepes, pH 7.5, 50 mM KCl, and 1 mM MgCl2) with 0.5% digitonin and protease inhibitor (cOmplete EDTA-Free; Roche). Insoluble components were removed by centrifugation at 20,000 g. Primary antibodies preadsorbed to protein G-sepharose beads (GE Healthcare) were added to the cell extract, and the mixture was incubated for 2 h at 4°C. After centrifugation, beads were washed with lysis buffer supplemented with 0.1% digitonin before elution in denaturing gel loading buffer for SDS-PAGE electrophoresis and Western blot analysis.
Isolation of mRNA and quantitative mRNA analysis was performed as previously described (Jonassen et al., 2008 (link)) using the primers tabulated in