FIG 2.

Protein synthesis in HeLa and KO cell lines. (A) Western blot. HeLa, SAMD9^−/−, WDR6^−/−, and FTSJ1^−/− cells were infected with WT VACV or ΔC7K1 at a multiplicity of infection of 5 PFU/cell. At 8 h, lysates were prepared, and proteins were resolved by SDS-PAGE and then transferred to membranes. The blots were probed with primary antibodies to I3, D13, and A3, followed by secondary antibodies. Protein bands were visualized with an infrared imager. Inter, intermediate. (B) Expression of SAMD9. HeLa, SAMD9^−/−, WDR6^−/−, and FTSJ1^−/− cells were infected as described for panel A and analyzed by SDS-PAGE. Blots were probed with antibody to SAMD9. (C) Detection of newly synthesized proteins by incorporation of [³⁵S]methionine and cysteine. HeLa and SAMD9^−/− cells were infected with ΔC7K1 at a multiplicity of 3 PFU/cell and incubated with [³⁵S]methionine and cysteine for 15 min at the indicated times. The cells were then washed, lysed, and analyzed by SDS-PAGE. Radiolabeled proteins were detected with a biomolecular imager. HPI, hours postinfection.