For lysis of S2R+ and adherent HEK-293T cells, cells were washed once with ice-cold PBS and then lysed with lysis buffer (1% Triton X-100, 40 mM HEPES pH 7.4, 10 mM β-glycerol phosphate, 10 mM sodium pyrophosphate, 2.5 mM magnesium chloride) and 1 tablet of EDTA-free protease inhibitor (Roche) per 25 mL buffer. Lysates were clarified by centrifugation at 21,000 × g at 4 °C for 10 min. Dissected Drosophila tissues and whole flies were crushed physically utilizing a bead beater in Triton lysis buffer and processed as above.
For anti-FLAG, anti-HA, or anti-myc immunoprecipitations leading to Western blot analyses, either anti-FLAG M2 agarose beads (Millipore Sigma) or anti-HA or anti-myc-coupled magnetic beads (Thermo Fisher Scientific) were used. Beads were washed three times prior to use with Triton lysis buffer and were then incubated with the supernatant of each clarified lysate for 2 h at 4 °C. Following immunoprecipitation, beads were washed three times with Triton lysis buffer supplemented to contain 300 mM NaCl. Immunoprecipitated proteins were denatured by addition of SDS-PAGE sample buffer and boiling at 95 °C for 3 min and resolved by 8%, 10%, or 4–20% SDS-PAGE before analysis by immunoblotting. All antibodies were used at a 1:1000 dilution, except for the anti-dS6K antibody, which was used at a 1:10,000 dilution.
For anti-FLAG, anti-HA, or anti-myc immunoprecipitations leading to Western blot analyses, either anti-FLAG M2 agarose beads (Millipore Sigma) or anti-HA or anti-myc-coupled magnetic beads (Thermo Fisher Scientific) were used. Beads were washed three times prior to use with Triton lysis buffer and were then incubated with the supernatant of each clarified lysate for 2 h at 4 °C. Following immunoprecipitation, beads were washed three times with Triton lysis buffer supplemented to contain 300 mM NaCl. Immunoprecipitated proteins were denatured by addition of SDS-PAGE sample buffer and boiling at 95 °C for 3 min and resolved by 8%, 10%, or 4–20% SDS-PAGE before analysis by immunoblotting. All antibodies were used at a 1:1000 dilution, except for the anti-dS6K antibody, which was used at a 1:10,000 dilution.