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. 2007 Dec 1;21(23):3135–3148. doi: 10.1101/gad.1597707

Figure 4.

Figure 4.

hCaf1 binds to PABC domain of PABPC1 via Tob with PAM2 motifs. (A) HeLa cells were transfected with pFlag-PABPC1 and either pHA-CMV5 (lanes 1,3,5,7) or pHA-hCaf1 (lanes 2,4,6,8). The cell extracts were subjected to an immunoprecipitation assay using anti-HA antibody in the presence or absence of RNaseA. The immunoprecipitates (lanes 5–8) and the inputs (lanes 1–4, 10% of the amount immunoprecipitated) were analyzed by immunoblotting using the indicated antibodies. (B) Recombinant PABPC1 and GST-hCaf1 prepared from E. coli were subjected to a GST pull-down assay in the presence (lane 2) or absence (lane 1) of recombinant Tob (1–285). Immunoblot analyses were performed with the indicated antibodies. (C) COS-7 cells were transfected with pHA-hCaf1 and either pMEFlag (lanes 1,5), pMEFlag-Tob (lanes 2,6), pMEFlag-Tob (1–218) (lanes 3,7), or pMEFlag-Tob (110–345) (lanes 4,8). The cell extracts were subjected to an immunoprecipitation assay using anti-Flag antibody. The immunoprecipitates (lanes 5–8) and the inputs (lanes 1–4, 20% of the amount immunoprecipitated) were analyzed by immunoblotting using the indicated antibodies. (D) COS-7 cells were transfected with pHA-hCaf1 and either pMEFlag (lanes 1,6), pMEFlag-Tob (lanes 2,7), pMEFlag-Tob F139A (lanes 3,8), pMEFlag-Tob F274A (lanes 4,9), or pMEFlag-Tob F139A/F274A (lanes 5,10). The cell extracts were subjected to an immunoprecipitation assay and immunoblot analyses as described in C. (Top) Tob protein is schematically represented. Gray bars indicate putative PAM2 motifs. The sequences of the two putative PAM2 motifs are also shown. The amino acid residues that were mutated in the experiment are shaded. (E) COS-7 cells were transfected with pMEMyc- Tob and either pFlag-PABPC1 (lanes 2,7), pFlag--PABPC1 F567A (lanes 3,8), pFlag-PABPC1 K580A/L585A (lanes 4,9), or pFlag-PABPC1 F567A/K580A/L585A (lane 5,10). (Lanes 1,6) As a control, COS-7 cells were transfected with pMEMyc and pFlag-PABPC1. The cell extracts were subjected to an immunoprecipitation assay using anti-Myc antibody. The immunoprecipitates (lanes 6–10) and the inputs (lanes 1–5, 5% of the amount immunoprecipitated) were analyzed by immunoblotting using the indicated antibodies.