FIGURE 9.

Differential regulation of Paip2A and Paip2B protein levels. (A) HEK 293T cells were transiently transfected by using increasing amounts of plasmids encoding HA-Paip2A or HA-Paip2B, as indicated. At 24 h post-transfection, cells were lysed and lysates were analyzed by Western blot using anti-HA. Samples from two independent experiments were loaded. (B) HEK 293T cells were transfected with plasmids encoding HA-tagged proteins (PABP, Paip2A, or Paip2B) and metabolically labeled for 2 h with [³⁵S]methionine and chased with cold methionine for 5 h in the absence or presence of 50 μM LLnL (proteasome inhibitor). Cell lysates were subjected to immunoprecipitation with anti-HA antibody, and immune complexes were washed and resolved by SDS-PAGE gels, dried, and exposed to X-ray film. (C) The same procedure as in B, but cells were metabolically labeled for 1 h with [³⁵S]methionine in the absence or presence of 10 μM MG132 (proteasome inhibitor) and were chased or not chased with cold methionine for 5 h, in the same conditions. After immunoprecipitation with anti-HA antibody, immune complexes were first washed and proteins resolved by SDS-PAGE gels and then transferred to nitrocellulose membranes and exposed to X-ray film (upper panel). After autoradiography membranes were analyzed by Western blot using anti-HA and anti-PABP antibodies (lower panels) as indicated. Numbers under the upper autoradiography panel represent the intensity of bands in each lane. For comparison, the values obtained at chase time 0 in the absence of MG132 were taken as 100%. The numbers between brackets were obtained when the values at chase time 0 in the presence of MG132 were taken as 100%. (D) Cells were cotransfected with plasmids encoding either HA-Paip2A or HA-Paip2B and a plasmid encoding His₆-ubiquitin. At the indicated times before lysis, some of the cells were treated with 50 μM LLnL. Cells were lysed in guanidine-HCl denaturing buffer, and His₆-tagged complexes were purified by using a metal affinity resin as described in the Materials and Methods. Purified proteins were eluted from the resin with 1× Laemmli sample buffer, and the presence of HA-Paip2A or HA-Paip2B was revealed by Western blotting with anti-HA (upper panel). Aliquots of cell lysates were also analyzed in the same way for the presence of HA-Paip2A and HA-Paip2B. (E) The same procedure as in D (upper panel), but cells were also transfected with an empty plasmid or with plasmids encoding either untagged Paip2A or Paip2B in combination with the His₆-ubiquitin plasmid. Half of the cells were treated with LLnL for 6 h before lysis. Proteins were visualized by Western blot using appropriate specific antisera for Paip2A and Paip2B proteins.