FIG 2.

ORF45 mediates c-Fos accumulation and phosphorylation. (A) Small amounts (10 ng/well) of wild-type (W) and double-mutated (M) c-Fos expression plasmids were cotransfected into 293T cells in 12-well plates with 100 ng/each of ORF45 and wild-type and kinase-dead (D) RSK2 and ERK constructs. Thirty-six hours later, the cells were extracted, and c-Fos accumulation was analyzed as indicated. (B) Large amounts (500 ng/well) of wild-type c-Fos construct were cotransfected as described above, and c-Fos phosphorylation was analyzed as indicated. (C) Wild-type Bac16 and ORF45-null STOP45-harboring iSLK+ cells were left uninduced or were induced with 1 μg/ml Dox and 1 mM NaB for 48 h and analyzed as indicated. (D) Whole-cell extracts were obtained from wild-type Bac16-harboring iSLK+ cells induced with 1 μg/ml Dox and 1 mM NaB, and the phosphorylation of c-Fos was detected by Western blotting. (E) Total RNA was extracted from wild-type Bac16- and ORF45-null STOP45-harboring iSLK+ cells that were untreated or treated with 1 μg/ml Dox and 1 mM NaB for 48 h, and c-Fos mRNA was detected using reverse transcription–real-time PCR.