In vitro interaction between RGS2 and eIF2Bε. (A) Flag-tagged eIF2Bε was expressed in Sf9 cells in either its monomeric form or as part of the eIF2B holoprotein (i.e., ± eIF2Bα/eIF2Bβ/eIF2Bδ/eIF2Bγ). The precleared cell lysate was obtained as described in Materials and methods, and the indicated RGS protein was added to these lysates to a final concentration of 500 nM. The mixture was immunoprecipitated with anti-Flag antibody, and samples were run on SDS-PAGE and transferred. Membranes were probed with antihistidine antibody to identify coimmunoprecipitated RGS proteins (top) or anti-eIF2Bε to view immunoprecipitated eIF2Bε (bottom) from lysate. The blots shown are representative of at least three independent experiments. (B) Coimmunoprecipitation of endogenous RGS2 with endogenous eIF2Bε. UMR-106 osteoblast-like osteosarcoma cells were treated for 3 h with 100 µM forskolin to induce RGS2 expression. Vehicle control and forskolin-treated cell lysates (1 mg total protein) were incubated with protein A/G agarose beads either without (lanes 1 and 3) or with (lanes 2 and 4) anti-eIF2Bε antibody, or else were added directly to the gel (100 µg total protein; lanes 5 and 6). Protein A/G agarose beads were extensively washed, and the bound protein was removed by heating and added to the gels (lane 1–4). The SDS-PAGE gel was transferred to PVDF membrane and probed using anti-RGS2 antibody (top) and stripped and reprobed using the same eIF2Bε antibody used for immunoprecipitation (IP; bottom). The results shown are typical of three independent experiments. Asterisks denote nonspecific immunoreactive bands. IB, immunoblot. Molecular mass indicators are expressed in kilodaltons.