FIG. 4.
Association of PIT-2 with actin. (A and B) CHO-PIT-2V and TE671 cells were incubated at the indicated [Pi] for 2 h. Cytochalasin D (Cyt. D) (1 μg/ml) was added (+) or not added (−) 45 min prior to analysis. (A) Western blot with rabbit anti–PIT-2 serum (top panel; exposures: CHO-PIT-2V, 15 s, and TE671, 15 min) and MAb AC-40 (bottom panel; exposure: 15 s). (B) Immunoprecipitation (IP) with AC-40 or a nonrelevant MAb (IgG2a) followed by Western blotting with rabbit anti–PIT-2 serum. Signals corresponding to PIT-2 and IgG are indicated. Exposures: CHO-PIT-2V, 3 min, and TE671, 10 min. (C) Confocal immunofluorescence microscopy of CHO-PIT-2V cells treated with cytochalasin D. Top panels show P5D4-Cy3 staining (red); middle panels show phalloidin-FITC staining (green); and bottom panels show superimposed staining (yellow). Bar, 5 μm.