Figure 4. PCTK3 negatively regulates FAK and Src tyrosine kinase activities.

(a) PCTK3-knockdown cell lysates were subjected to immunoblot analysis using anti-FAK, anti-Src, anti-phospho-FAK (Tyr-397), anti-phospho-Src (Tyr-416), anti-phospho-Src (Tyr-527), and anti-PCTK3 antibodies. Immunoblot intensity of GAPDH was used as a loading control. Relative phosphorylation of FAK and Src were normalized by total FAK and Src levels, respectively. The phosphorylation of FAK and Src in NC siRNA cells was taken as 1. Results are expressed as means ± S.E. from three independent experiments. Statistical significance was determined by Student’s t-test. *p < 0.05 and **p < 0.01. (b) HEK293T cells plated on poly-L-lysine-coated plates were transfected with negative control (NC) siRNA or PCTK3 siRNA. Twenty-four hours later, cells were transfected with Halo-tagged FAK1 Y397F mutant for 24 hours. The cell lysates were subjected to immunoblot analysis using anti-phospho-cofilin (Ser-3) antibody. Relative phosphorylation of cofilin at Ser-3 was normalized by total cofilin levels. The phosphorylation of cofilin at Ser-3 in NC siRNA-transfected cells were taken as 1. (c–e) HEK293T cells were plated on poly-L-lysine-coated coverslips. Cells were transfected with NC or PCTK3 siRNA for 48 hours, fixed, and immunostained with anti-FAK (c), anti-phospho-FAK (Tyr-397) (d), and anti-phospho-Src family (Tyr-416) (e) antibodies, and Alexa 555-conjugated phalloidin (Red) (d,e). Hoechst nuclear staining is represented in blue. All images were obtained using a 63× objective lens. The digitally 4–6 times magnified images of different field-of-views are shown in the lower panel.