Figure 1.

Arc3 associates with the Arp2/3 complex in a F-actin dependent manner. (A) Lysates from cells expressing Arc3-MYC were prepared for immunoprecipitation (IP) — antibodies used are as indicated (top) and purified mouse IgG was used as a control. Immunoprecipitated proteins were analyzed by Western blots using antibodies for the indicated proteins. (B) The binding between Arc3 and Arc4 was determined by the yeast two-hybrid system by measuring the activation of the HIS3 reporter gene as described (Chang, et al., 1994). pVJLARC3 and pGADARC4 express Arc3 and Arc4 fused to the LexA DNA binding domain (LBD) and the Gal4 activation domain (GAD), respectively. (C) Cells expressing Arc3-GFP were fixed and stained with rhodamine-conjugated phalloidin to visualize F-actin. We found that nearly every GFP dot overlaps with an F-actin dot/patch, but not with F-actin cables, in all the cells that we examined. A representative cell is shown. (D) Arc3-GFP expressing cells were treated with 10 µM Latrunculin A (LatA) for 30 mins to depolymerize F-actin followed by washing and incubation in regular media (Wash out). Images of Arc3-GFP distribution were collected from untreated cells, from cells treated with LatA for 30 mins and from cells 1 hour after washing out. Bar, 1 µm.