FIG 4.

CPj0678 recruits PACSIN2 on the plasma membrane through its C terminus. (A) The purified GST or GST–CPj0678-C protein and the total protein extracted from HEp-2 cells were mixed and precipitated with glutathione-Sepharose 4B beads. Eluates from the beads were subjected to SDS-PAGE and analyzed by mass spectrometry. Arrowhead indicates a band at a molecular size of approximately 60 kDa. (B) GFP or CPj0678-GFP was transfected into HEp-2 cells. Cells were lysed in lysis buffer, and the expression of PACSIN2, GFP, and GFP-CPj0678 were determined by immunoblotting with specific antibodies. The extracted protein was immunoprecipitated with anti-GFP antibodies conjugated with protein A beads and analyzed by immunoblotting using anti-PACSIN2 antibodies. The experiments were performed in triplicate. (C) CPj0678-HA and DMT1A-I were cotransfected into HeLa cells. Cells were fixed and stained with mouse anti-HA antibodies. Thin scale bar = 10 μm, and thick scale bar = 5 μm. (D) PACSIN2-GFP alone, CPj0678-mCherry alone, or both PACSIN2-GFP and CPj0678 were transfected into HEp-2 cells. Cells were fixed with 4% paraformaldehyde (PFA), and nuclei were stained with Hoechst33342. The representative image is from experiments performed in triplicate.