Fig. 3. Thr1590 plays an essential role in the effects of Kalirin-7 on PC12 cell morphology. (A) PC12 cells expressing Kalirin-7 T/A or Kalirin-7 T/D were examined 24 hours post-transfection. Kalirin-7 was visualized using antibody to Myc (green) and filamentous actin was visualized using TRITC-phalloidin (red). Arrowhead indicates protrusion, stars indicate nontransfected cells, arrows indicate nuclei. (B) Cell perimeter and roundness were quantified as described in Fig. 1: GFP, n=27; Kalirin-7 (Kal7), n=24; Kalirin-7-T/A (Kal7 T/A), n=15; Kalirin-7-T/D (Kal7 T/D), n=24. (C) PC12 cells cotransfected with vectors encoding GFP-DN Cdk5 and either Kalirin-7 T/A or Kalirin-7 T/D were fixed after 24 hours. Exogenous Kalirin was visualized with Myc antibody (blue); DN Cdk5 was identified based on coexpression of GFP; filamentous actin was visualized with TRITC-phalloidin (red). Arrowhead, protrusion; white stars, nontransfected cells; arrows, nuclei. (D) Cell perimeter and cell roundness were evaluated as described in Fig. 1: control GFP, n=18; Kalirin-7-T/D, n=15; Kalirin-7-T/D with DN Cdk5, n=18. P values were calculated using Student's t-test (two-tailed) and bars indicate s.e.m.; ^#P<0.001; ^*P<0.05. Scale bars, 10 µm.