α flag beads

Drosophila S2 cells were transiently transfected with pAc-brm-FLAG-6xHIS in combination with either pAc-clk-V5-HIS or pAc-V5-HIS empty plasmid using Effectene (Qiagen). For cycloheximide experiments, protein was extracted with EB2 (recipe is listed in “Protein extraction from Drosophila S2 cells and fly heads” section), and CHX was added to a final concentration of 10μg/ml 48 hours post-transfection. Cells were harvested every 2 hours over a 6-hour period after CHX addition. SDS-PAGE and Western blotting and detection were performed as described in the “Western blotting of protein extracts, detection, and quantification” section. For λ_pp experiments, protein was extracted with EB2 supplemented with PhosStop (Roche, Indianapolis, IN) and were subjected to IP with 15 μl of settled α-FLAG beads (Sigma) per reaction for 4 hours at 4°C. Beads were washed 2 times with EB2 without NaF or PhosStop and one time with λ_pp buffer (New England Biolabs, Ipswich, MA) before resuspension in 40 μl of λ_pp buffer. Experimental reactions were treated with 0.6ul λ_pp (New England Biolabs), and both experimental and control reactions were then incubated in a 30°C water bath for 30 mins. 45 μl of 2X SDS sample buffer was added to the beads for protein elution. Eluted protein was subjected to SDS-PAGE and Western blotting and detection. CHX and λ_pp experiments were each performed 3 times.