Figure 5.

Phosphorylated Coronin 1B associates with WISp39 and SSH. (A) Coronin 1B (Coro 1B) associates with WISp39 and SSH when it is phosphorylated. HEK293 cells were transfected with GFP-tagged WT, S2A, or S2D Coronin 1B. Lysates were immunoprecipitated with anti-GFP antibody and analyzed by Western blotting with indicated antibodies. The experiments were repeated three times with similar results. (B) Coronin 1B associates with SSH only in the presence of WISp39. U2OS cells were cotransfected with control or WISp39 siRNA and GFP-tagged WT Coronin 1B. Lysates were immunoprecipitated with anti-GFP antibody and analyzed for association with SSH by Western blotting. The experiments were repeated more than three times with similar results. (C) Coronin 1B phosphorylation increases in the absence of WISp39. HeLa cells were cotransfected with control or WISp39 siRNA and HA-tagged WT Coronin 1B. Lysates were immunoprecipitated with the anti-HA antibody (HA–Coronin 1B) and analyzed for phosphorylation of Coronin 1B on Ser2 by Western blotting with phospho-PKC substrate-specific antibody (phospho-Ser). Quantitation of the phospho-Ser Coronin 1B in control and WISp39 KD cells from three experiments is shown. Student’s t test; **, P ≤ 0.01. (D) WISp39 KD increases phosphorylated Cofilin. U2OS cells were transfected with control or WISp39 siRNA, and the cell lysates were analyzed for phosphorylation by Western blotting with antibodies recognizing phospho-Cofilin. Quantitation of the phospho-Cofilin in control and WISp39 KD cells from three experiments is shown. Student’s t test; **, P ≤ 0.01. (E) WISp39 KD does not influence the levels of other motility-associated proteins. U2OS cells were transfected with control or WISp39 siRNA, and the cell lysates were analyzed by Western blotting with the indicated antibodies. Ctrl, control; IP, immunoprecipitation.