FIGURE 3:

Mena–GRASP65 interaction is required for Golgi integrity. (A) BSA- or GRASP65-coated beads were incubated with purified GST-Mena EVH1, washed, and analyzed by Western blot for GST. (B) Beads coupled with BSA or purified GRASP65 aa 1–201 (p65N) or 202–446 (p65C) fragment were incubated with purified His-Mena EVH1, washed, and analyzed by Western blot for His tag. (C) Beads coupled with GST or purified GST-tagged GRASP65 fragments were incubated with HeLa cell lysate, washed, and analyzed by Western blot for Mena. (D) GRASP65 point mutations, with indicated amino acids mutated. (E) Alignment of rat, mouse, and human GRASP65 amino acid sequences in the indicated region. Bold lines indicate the sites where conserved prolines were mutated in the mutants (M1–M6) as in D. (F) Cells transfected with GFP, GFP-tagged GRASP65 WT, or indicated mutants were immunoprecipitated with anti-GFP antibodies and analyzed by Western blot. (G–J) GRASP65-depleted cells were transfected with WT GRASP65 or its M2 mutant and analyzed by Western blot (G) and microscopy (H, I). Bar, 20 μm. (J) Quantitation of cells in I with fragmented Golgi. ***p < 0.001.