Western Blot Analysis of eIF2α Phosphorylation

Cells were grown in either 1% or 10% FCS as described in the “Cell culture” section. The western blot protocol was adapted from our previous work [58 (link)]. Cells were lysed in 300 μl RIPA buffer containing protease inhibitor and phosphatase inhibitor. Fifteen to 25 μg total protein was loaded on 10% SDS PAGE, and electrophoresis was performed for 1 h at 120 V to resolve the proteins. We followed the protocol from Cell Signaling for transferring, blocking, incubating, washing, and developing the membrane. Cells treated with 1 μM thapsigargin (TG) and 1 μM hydrogen peroxide for 5 h were also included as a positive control for phosphorylation of eIf2α protein. Tubulin was used as a loading control. The following antibodies were used in the western blot analysis: Phospho-eIF2α (Ser51) Antibody (Cell Signaling #9721), eIF2 α Antibody (Cell Signaling #9722), and α-Tubulin Antibody (Merck Millipore #CP06).
For quantification, band intensities of eIF2α and p-eIF2α were normalized by the corresponding loading control (tubulin). Then, for both eIF2α and p-eIF2α, normalized band intensities were divided by the average normalized band intensity across the different conditions. Finally, the ratio of p-eIF2α to eIF2α was calculated by dividing the normalized intensities calculated in the previous step. The band intensities were quantified using the ImageJ software [60 (link)].