Cst cell lysis buffer

Following the organelle fractionation protocol with or without 2 h serum starvation, cells were washed with cold PEE, suspended in Bud Buffer, and cracked using a Balch homogenizer. Fractions obtained after organelle fractionation were thoroughly mixed and divided into equal volumes before adding beads and/or primary antibody. Primary antibody and 10× Cell Signaling Technology (CST) cell lysis buffer (CST to 1× final concentration) were added to each IP sample. Samples were incubated, rotating, at 4°C overnight. A 10% bead solution (Protein A/G UltraLink Resin, Thermo Fisher #53132) was prepared in block buffer (1× lysis buffer containing 5% BSA). Beads were rotated 1 h at 4°C and washed twice with block. Beads were resuspended in 1 mg/ml BSA in 1× lysis buffer. Bead solution was added to each sample and incubated, rotating, at 4°C for 2 h. After incubation, IP beads were washed four times with 1× lysis buffer containing decreasing amounts of BSA (two washes with 0.25 mg/ml, followed by 0.1 mg/ml, and finally no BSA). Samples were resuspended in 7 M Urea sample buffer with DTT before SDS–PAGE and blotted on nitrocellulose.