Elucidating eIF2α-PHLPP Interaction

Cells were harvested and detergent-solubilized cell lysates were obtained as described previously [5 (link), 7 (link), 8 (link), 17 (link)]. Equal amounts of cell lysates were resolved by SDS-PAGE and subjected to western blot analysis. To examine the interaction between eIF2α and PHLPP, the solubilized cell lysates were incubated with either the anti-HA high-affinity antibody or the anti-PHLPP1 or PHLPP2 antibodies and protein A/G agarose beads (Thermo) at 4 °C for overnight. The beads were washed three times with lysis buffer and the immunoprecipitated proteins were analyzed by SDS-PAGE and Western blotting. The phospho-eIF2α, total eIF2α, ATF4, and LC3 antibodies were obtained from Cell Signaling. The PHLPP1 and PHLPP2 antibodies were from Bethyl laboratories. The β-actin and the anti-HA high-affinity antibodies were from MilliporeSigma.