FIG. 3.

Stable overexpression of RACK1 in fibroblasts results in its altered subcellular localization. (A) NIH 3T3-IGFR cells were cotransfected with pBabePuro and phEF-RACK1-HA or phEF-Neo and selected for puromycin resistance as described in Materials and Methods. RACK1-transfected clonal cell lines (clones 1, 4, 9, and 20), a culture composed of pooled RACK1-transfected clones (P), and the empty vector-transfected pooled culture (C) were established. Portions (10 μg) of RIPA-extracted total cell lysate protein from each cell line were fractionated by SDS-12% PAGE and transferred to a membrane which was cut in half and immunoblotted (IB) with anti-RACK1 (lower half) or anti-IGFR (upper half) Ab. Arrows indicate endogenous and exogenous (transfected) RACK1. (B) NIH 3T3-IGFR control (C) and RACK1-overexpressing cells (clones 1 and 4) were separated into Triton X-100-insoluble (i) and Triton X-100-soluble (s) fractions as described in Materials and Methods. Then, 10 μl of each fraction was separated in an SDS-12% polyacrylamide gel, transferred to a membrane, and immunoblotted with anti-RACK1 Ab. (C) Serum-starved control (C) and RACK1-overexpressing (clones 1 and 4) NIH 3T3-IGFR cells were mock treated or treated with IGF-I for 10 min. Cell lysates were prepared with 1% digitonin buffer and 1 mg of cell lysate was immunoprecipitated (IP) with anti-RACK1 Ab, resolved in an SDS-12% polyacrylamide gel, and transferred to a membrane which was cut in half and immunoblotted with anti-IGFR (upper half) or anti-RACK1 (lower half) Ab.