Quick western kit

Cells were collected in lysis buffer (1 mM EGTA, 1 mM EDTA, 10 mM Tris [pH 7.4], 150 mM sodium chloride, 1% deoxycholate, 1% NP-40, and supplemented with protease and phosphatase inhibitor cocktail tablets [cOmplete mini EDTA-free and PhosStop; Roche]). Lysates were precleared with 10 μl of protein A/G PLUS Sepharose Beads (Santa Cruz Biotechnology) for 1 h at 4°C. The precleared lysates were then incubated with 20 μl of protein A/G PLUS Sepharose Beads, which had previously been coupled with the appropriate primary antibody for 2 h at 4°C on an orbital shaker. The beads were washed three times with lysis buffer supplemented with protease and phosphatase inhibitors. The immunoprecipitation complexes were eluted from the beads by boiling in 2× SDS buffer for 5 min.
For Western blotting analyses, protein lysates/coimmunoprecipitation complexes were resolved on a 4%–12% gradient gel (Invitrogen) and blotted on a nitrocellulose membrane with the appropriate primary antibodies (1:1,000) and IRDye-conjugated anti-mouse, anti-goat, or anti-rabbit secondary antibodies or the Quick Western Kit (926-69100; LI-COR), as appropriate. Images were acquired on a LI-COR Odyssey infrared scanner. Densitometric quantification of bands was performed using Image Studio software (LI-COR).

Extraction of total protein extracts and analysis by Western immunoblotting and coIP was recently described in detail [66 (link)]. Proteins for coIP experiments were isolated 48 h after siRNA transfection. Antibodies used in this study are listed in Suppl. Table S2. Signal detection for coIP experiments was done using the Quick Western Kit (LI-COR Biosciences, Bad Homburg, Germany).

Cells were rinsed twice with PBS and lysed in NP-40 lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1% NP-40 and 2 mM EDTA), then sonicated for 15 min on ice. The soluble fraction of the lysate was collected by centrifugation at 16,000 x g for 10 min prior to immunoprecipitation as described above. For immunoprecipitations, the primary antibodies were added to the lysate and incubated on a rotator overnight at 4°C. The immune complexes were then pulled down with protein A/G magnetic beads (ThermoFisher, Cat. # 88803) and washed three times with washing buffer according to the manufacturer’s manual. The immunoprecipitates were eluted by incubating the beads with SDS-PAGE loading dye for 10 min at room temperature and boiled for 10 min at 95°C prior to being loaded on the SDS-PAGE gel. Western blotting was performed as described above. For immunoprecipitation of endogenous proteins, western blotting was performed with the quick western kit (Li-Cor, Cat. # 926–69100) according to the manufacturer’s instructions.