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. 2004 Mar;24(5):2025–2040. doi: 10.1128/MCB.24.5.2025-2040.2004

FIG. 1.

FIG. 1.

(A) Inducible expression of eIF2α variants in 3T3 L1 cells. Cells carrying vector alone or tetracycline-inducible plasmids expressing empty vector, FLAG eIF2α-S51D, or FLAG eIF2α-S51A were induced by the withdrawal of DOX, and whole-cell lysates were harvested at 0, 3, and 6 days postinduction. Cell extracts were analyzed for the expression of FLAG-tagged proteins by immunoblotting with a MAb directed against the FLAG epitope as described in Materials and Methods. Lane 1 is whole-cell lysate harvested from 293T cells transiently transfected for 48 h with a plasmid expressing FLAG-tagged WT eIF2α. (B) Morphological changes induced by expression of eIF2α variants. The vector-, FLAG eIF2α-S51D-, and FLAG eIF2α-S51A-expressing cell lines were induced to express the indicated variants for 6 days, and cell morphology was monitored at 0 and 6 days postinduction by phase-contrast microscopy at ×4 and ×20 magnifications. (C) Levels of total and phosphorylated eIF2α in DOX-inducible cell lines. Whole-cell lysates from 3T3 L1 DOX-inducible cells either uninduced or induced to express the indicated eIF2α variants for 6 days were harvested, electrophoresed, and probed with MAbs directed against either total eIF2α or eIF2α specifically phosphorylated on serine 51.