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. 2002 Aug;76(15):7874–7882. doi: 10.1128/JVI.76.15.7874-7882.2002

FIG. 4.

FIG. 4.

Proteins that bind to Purv in mock- or HSV-infected Vero or HEL cells. Protein extracts of mock- (M) or HSV-infected (H) Vero and HEL cells were incubated with Purv (P) or Me-Purv (Me) covalently linked to agarose beads. Purv-binding proteins were eluted, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and silver stained or immunoblotted with specific antibodies (see text for descriptions of antibodies used). The antibodies specific for cdk4, -6, and -9 did not recognize simian cdk4, -6, or -9 with high affinity and, thus, could not be used in Vero cells. (A) Vero cells were infected with wild-type HSV-1 and treated with the concentrations of drug indicated on the x axis. Viral titers at 20 h postinfection are plotted against concentrations of Me-Purv (▴), Purv (▵), or Rosco (□). (B) Silver-stained Purv-binding proteins in extracts of mock- (M) or HSV (H)-infected Vero and HEL cells. Molecular masses are indicated on the left in kilodaltons, and the ranges of molecular masses of the cdks targeted by Purv in vitro and their corresponding cyclins are indicated by the brackets on the right. No Vero cell protein bound to Me-Purv was detected (data not shown). Proteins in HEL cell extracts that bound to Purv, and not to Me-Purv, are indicated by white arrowheads on the right side of the gel. (C) Western blots of Purv-binding proteins in mock- (M)or HSV (H)-infected Vero cell extracts. Purv-binding proteins (P) or total proteins in cell extracts (Extract) were resolved by SDS-PAGE, transferred, and blotted with antibodies specific for HSV-1 proteins. Molecular masses are indicated on the left in kilodaltons. (D) Western blots of Purv- (P) or Me-Purv (Me)-binding proteins in mock- (M) or HSV (H)-infected Vero cell extracts. Purv- or Me-Purv-binding proteins or total proteins in cell extracts (Extract) were resolved by SDS-PAGE, transferred, and blotted with antibodies specific for cdk7, -5, -2, and -1 or erk2 as indicated on the right of each blot. Only the relevant sections of the blots are shown. Exposure times differed for each antibody. (E) Western blots of Purv- (P) or Me-Purv (Me)-binding proteins in mock- (M) or HSV (H)-infected HEL cell extracts. Purv- or Me-Purv-binding proteins or total proteins in cell extracts were resolved by SDS-PAGE, transferred, and blotted with antibodies specific for cdk7, -5, and -2, erk1 and -2, and cdk9, -6, -4, or -1 as indicated on the right side of each blot.