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. 2007 Jan 8;27(6):2324–2342. doi: 10.1128/MCB.02300-06

FIG. 9.

FIG. 9.

Overexpression of Caprin-1 induces of phosphorylation of eIF-2α through a mechanism that depends on RNA binding. (A) Overexpression of Caprin-1 induced phosphorylation of eIF-2α. 293T cells were transfected with vector alone or Flag-tagged Caprin-1. After 48 h, the total cell lysates were separated on an SDS-PAGE gel and immunoblotted for phosphorylated eIF-2α, for total eIF-2α, and for β-actin as a loading control. (B) Caprin-1 induces eIF-2α phosphorylation through an RNA-dependent mechanism. As in panel A, 293T cells were transfected with Flag-tagged Caprin-1 or Flag-tagged Caprin-1 mutants and, for controls, with Flag-smgGDS or empty vector and incubated for 48 h prior to lysis. As a positive control for eIF-2α phosphorylation, 293T cells were stressed with 0.5 mM sodium arsenite for 1 h. Total cell lysates were separated on an SDS-PAGE gel and immunoblotted for phosphorylated eIF-2α and total eIF-2α. The expression of the Flag-tagged proteins was confirmed by immunoblotting with anti-Flag antibodies.