Preparation of HEK cell lysates for Pull-down and Western Blot (WB)

HEK cells were harvested 16-24 hours after transfection by vigorous pipetting. Cell suspension was centrifuged at 700g’ for 1 min, then washed once with cold PBS and spun again. For direct WB analysis of cell lysate, pellet was lysed in RIPA buffer: NaCl 150mM, TX-100 1%, sodium deoxycholate (SDC) 0.5% and sodium dodecyl sulfate (SDS) 0.1%, Tris 50mM, pH 8.0 supplemented with protease and phosphatase Inhibitors. For pull-down experiments, a milder detergent buffer was used instead: NaCl 150mM, TX-100 1% and Tris 25mM pH 8.0. Cells were mixed 10x by pipetting and incubated in the lysis buffer for 5 minutes on ice then centrifuged 1000xg’ for 5’ at 4°C. Cleared supernatant lysate was collected into a fresh tube and used for pull-down or direct analysis. For SDS-PAGE, samples were mixed 1:3 with Sample Buffer x4 (Tris 500mM pH 6.8, SDS 8%, glycerol 2.5%, DTT 10mM and bromophenol Blue), then heated to 95°C for 5’.