Figure 5.

UL135 Interacts Independently with the WRC and Talin and Inhibits Cytoskeletal Remodelling

(A–E) HFFF-hCAR were transfected with control siRNA (siRNA-Ctrl), siRNA against ABI1 and ABI2 (siRNA-ABI1/ABI2), siRNA against talin-1 and talin-2 (siRNA-TLN1/2), or siRNA against CYFIP1. They were infected with RAd-UL135 or RAd-Ctrl 24 hr later. Assays were performed 48 hr postinfection.

(A and B) Cells were fixed and stained for actin (phalloidin).

(C and D) Cells were allowed to adhere to fibronectin-coated dishes for 30 min and then fixed, and the number of cells exhibiting a spread morphology was counted by microscopy. Four separate fields were counted; results are mean ± SD and representative of three independent experiments. Two-way ANOVA test. ^∗∗∗p < 0.001.

(E) Cells were lysed, and UL135 was immunoprecipitated with its V5 tag. Immunoprecipitated proteins were separated by SDS-PAGE, and western blot was performed for the indicated proteins.

(F–J) HFFF-hCAR were infected with the indicated adenovirus vectors, and assays were performed at 48 hr postinfection.

(F) UL135 was immunoprecipitated with the V5 tag. Immunoprecipitated proteins were separated by SDS-PAGE, and western blot was performed for the indicated proteins.

(G) Cells were fixed and stained for actin (phalloidin).

(H) Cells were fixed and stained for UL135 (V5 antibody) and ABI1. Accumulation of ABI1 at sites of actin protrusions are indicated with arrows.

(I) Cells were stained for UL135 (V5 antibody) and talin. Accumulation of talin at sites resembling focal adhesions are indicated with arrows.

(J) Cells were allowed to adhere to fibronectin-coated dishes for 30 min and then fixed, and the number of cells exhibiting a spread morphology was counted by microscopy. Four separate fields were counted; results are mean ± SD and are representative of three independent experiments. One-way ANOVA test. ^∗∗∗p < 0.001.