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. 1999 Jul;19(7):4611–4622. doi: 10.1128/mcb.19.7.4611

FIG. 3.

FIG. 3

Formation of GRF1 and GRF2 oligomers in mammalian cells. (A) GRF oligomerization occurs in NIH 3T3 cells between GST-GRF1 and wild-type GRF1 or wild-type GRF2, but it is deficient between GST-GRF1 and the L263Q GRF1 mutant. Metabolically labeled NIH 3T3 cells transiently expressed mGRF1, mGRF1L263Q, and hGRF2, with or without GST-mGRF1 or GST, as indicated. Cell lysates were immunoprecipitated with antisera to GRF1 (lanes 1 and 2), GRF2 (lane 3) or precipitated with glutathione-Sepharose (GS) beads (lanes 4 to 7). The arrows indicate the location of GRF1 (lanes 4 and 5) or GRF2 (lane 6) coprecipitated with GST-GRF1 (lanes 4 to 6) and the location of GST (lane 7). (B) Wild-type GRF1 and the DH cluster mutant form oligomers in 293T cells, but oligomerization is deficient with the L263Q mutant. Transiently transfected cells expressing wild-type (lanes 1, 2, 4, and 6) or mutant GRF1 (L263Q in lanes 3 and 5; cluster mutant [GRF1*] in lane 7), with or without coexpression of GST-GRF1, were precipitated by glutathione-Sepharose (GS) beads or GRF antibodies as indicated, followed by anti-GRF1 blotting. The designated oligomerized proteins are marked with arrows. (C) Oligomerization in 293T cells. In lanes 1 to 3, lysates from cells expressing GRF2 with or without coexpression of GST-GRF1 or GST were immunoprecipitated and blotted with anti-GRF2. In lanes 4 and 5, lysates from cells coexpressing GST-GRF2 and GRF1 or GRF2 were precipitated with glutathione-Sepharose (GS) beads followed by anti-GRF2 blotting. In the bottom panels, the anti-GRF2 blots were (incompletely) stripped and were reprobed with GRF1 antibodies. The designated oligomerized proteins are marked with arrows. (D) A rat brain extract (RBE) contains GRF1-GRF2 oligomers. Lanes: 1 and 2, extracts from 293T cells expressing GRF2 and GRF1, respectively, Western blotted with anti-GRF2 antibodies; 3 and 4, rat brain extracts immunoprecipitated with anti-GRF1 and anti-GRF2, respectively; 5, 293T cells coexpressing GRF1 and GRF2 immunoprecipitated with anti-GRF2 antibodies; 6 and 7, 293T cells expressing GRF1 immunoprecipitated with anti-GRF1 or anti-GRF2 antibodies, respectively.