FIG. 1.

IGF-I and PMA induce the intracellular association of RACK1, IGF-IR, and PKC. HEK293T cells transiently transfected with phEF-IGFR, NIH 3T3-IGFR cells, and Rat2-IGFR cells were serum starved overnight and either mock treated or treated with IGF-I or PMA for 10 min. Cell lysates were prepared with digitonin buffer. (A) A 1-mg portion of total cell lysate was immunoprecipitated (IP) with anti-RACK1 or anti-IGFR Ab (HEK293T and NIH 3T3-IGFR). Immunoprecipitates were fractionated by SDS-12% PAGE and transferred to a nitrocellulose membrane. The membrane was cut in half: the upper half was immunoblotted (IB) with anti-IGFR Ab (top panels), and the lower half was immunoblotted with anti-RACK1 Ab (middle panels). The membranes containing RACK1 (middle panel in the RACK1 IP) or IGF-IR (top panel in the IGFR IP) were stripped and reprobed with anti-phosphotyrosine Ab (anti-pTyr) (bottom panels). (B) A 1-mg portion of total cell lysate was immunoprecipitated with anti-PKCμ (HEK293T), anti-PKCδ (HEK293T), or anti-RACK1 (Rat2-IGFR) Ab. Immunoprecipitates were separated by SDS-12% PAGE and transferred to a membrane which was cut in half: the lower half was immunoblotted with anti-RACK1 (top panels of HEK293T), and the upper half was immunoblotted with anti-IGFR (bottom panels of HEK293T) or anti-PKCμ (top panels of Rat2-IGFR) Ab as shown.