FIGURE 6.

Characterization of the different HAP1 cell lines by immunocytochemistry and western blotting. (A) HAP1 WT, HAP1-eIF2A^-, HAP1-eIF2D^-, and HAP1-eIF2A^-/eIF2D^- cells were seeded on microscope coverslips, fixed and stained with primary rabbit polyclonal anti-eIF2A or anti-eIF2D antibodies and a mouse monoclonal anti-eIF2α antibody. An anti-mouse antibody conjugated to Alexa 555 was used to detect eIF2α (red) and an anti-rabbit antibody conjugated to Alexa 488 was employed to detect eIF2A and eIF2D (green). DAPI was used to stain the nuclei (blue). Scale bar, 20 μm. (B) The presence of eIF2A or eIF2D in HAP1 WT, HAP1-eIF2A^-, HAP1-eIF2D^-, and HAP1-eIF2A^-/eIF2D^- cells was also determined by western blotting with rabbit polyclonal anti-eIF2A and anti-eIF2D antibodies. eIF2α was used as loading control for the four cell lines. Proteins were resolved by SDS-PAGE and samples were probed with antibodies to show the presence of these factors in the HAP1 cells lines. Shown is a representative blot from three independent experiments.