FIGURE 8.

Characterization of an antibody specific for Paip2B: tissue-specific distribution of Paip2 proteins. A Paip2B-specific antibody was obtained by immunizing rabbits with GST-Paip2B (residues 1–21). (A) 293T cells were transfected with plasmids encoding HA-Paip2A or HA-Paip2B, and cell extracts were analyzed by Western blot using anti-Paip2B and anti-HA. (B) 293T cells were transfected with plasmids containing either no insert (negative control) or encoding either HA-Paip2A or HA-Paip2B. Cell extracts were subjected to immunoprecipitation using anti-Paip2A or anti-Paip2B, as indicated. The immune complexes were analyzed by far-Western blotting using ³²P-HMK-PABP as a probe. The electrophoretic migration of endogenous and HA-tagged Paip2 proteins is indicated. (C) Equal amounts of total protein (150 μg) from mouse tissue extracts were subjected to SDS-PAGE and analyzed by Western blot using antibodies against Paip2B, Paip2A, PABP, or eIF2α (as a loading control), as indicated, and by far-Western blot using a ³²P-HMK-PABP probe (bottom panel). The values under the Western blot panels represent the intensities of bands in each tissue normalized with respect to the corresponding eIF2alpha bands. For comparison, the value obtained for the small intestine sample was set as one. (D) 293T, HeLa, COS-7, and NIH 3T3 cell extracts were prepared, and equal amounts of protein were subjected either to immunoprecipitation using anti-Paip2B or anti-Paip2A antibodies or to SDS-PAGE (50 μg of whole-cell extract) and Western blotting using the same antibodies. The immune complexes were analyzed by far-Western blotting using a ³²P-HMK-PABP probe.