FIG. 1.

Caprin-1 and G3BP-1 associate and colocalize in RNA-rich cytoplasmic granules. (A, B) G3BP-1 coprecipitates with Caprin-1. In panel A, 293T cells were transfected with vector alone or Flag-Caprin-1, and total lysates were subjected to anti-Flag IP. The eluate of the immunoprecipitate (IP) and a 1/10 aliquot of the supernatant remaining after IP (S/N) together with a 1/10 aliquot of the whole-cell lysate (WCL) were run in parallel on SDS-PAGE and blotted for endogenous G3BP-1 with a mouse MAb to G3BP-1. Note that the efficient precipitation of the Flag-Caprin-1 was accompanied by a clearing of G3BP-1 from the post-IP supernatant. Panel B shows a reciprocal experiment in which coprecipitation of endogenous Caprin-1 was detected with anti-Caprin-1 rabbit serum. (C) Colocalization of Caprin-1 and G3BP-1 in cytoplasmic RNA granules. Actively proliferating HeLa cells were stained for endogenous Caprin-1 (green) and G3BP-1 (red). Nuclei were stained with DAPI (blue). The arrows in the enlarged area shown in the inset show colocalization of Caprin-1 and G3BP-1 in cytoplasmic granules. (D) Colocalization of Caprin-1 and RNA in cytoplasmic granules. NIH 3T3 cells were fixed and stained for total cellular RNA using 1 μM ethidium bromide (red) according to the method of Tang et al. (44) and with rabbit anti-Caprin-1 serum (green). As a control, some slides were pretreated with RNase before staining with ethidium bromide and anti-Caprin-1 serum. Note the lack of ethidium bromide staining in the cytoplasmic granules and the nucleoli in the nucleus after RNase treatment.