Figure 3
Figure 3. Both interaction with PA28γ and PA28γ proteasomal activity are required for p30-mediated inhibition of tax/rex RNA. (A) p30 retains the ability to interact with exogenously expressed PA28γ in MEF PA28γ knockout cells. (B-C) p30-mediated repression of tax/rex RNA is reduced in MEF PA28γ−/− compared with MEF wild-type cells. p30 activity is rescued by exogenous expression of FLAG-PA28γ. (D) Characterization of FT-27 cells stably knocked-down for endogenous PA28γ. Western blots show drastically reduced PA28γ expression, whereas expression of related PA28α and PA28β were not affected by shRNA used. (E) p30 efficiently inhibits tax/rex mRNA in 293FT cells but not in FT-27 knocked down for PA28γ expression. Lack of p30-mediated repression was rescued by ectopic expression of a PA28γ expression vector. (F) In contrast to PA28γ, a PA28γ mutant defective for proteasome activation N151Y cannot rescue p30-mediated repression of tax/rex RNA. PA28γ (α21-26), unable to bind p30, was not able to rescue p30-mediated repression of tax/rex RNA. Western blots show p30 and PA28γ expression in transfected cells. (G) FLAG-PA28γ was transfected into 293T cells in the presence or absence of p30-HA or p28-HA expression vectors. Cell lysates were used for immunoprecipitations as indicated. (H) Repressive effects of p30 and p28 were analyzed by transfection of 293T cells with HTLV-1 molecular clone pBST, HTLV-1-LTR-luciferase reporter and either p30HA or p28HA as indicated. Western blots were performed to show relative levels of p30 and p28 expression. Results are representative of 3 independent experiments. (I) Repressive effects of p30 and p28 were analyzed by transfection of 293T cells with HTLV-2 molecular clone SV2neoHTLV-2, HTLV-1-LTR-luciferase reporter and either p30HA or p28HA as indicated. Western blots were performed to show relative levels of p30 and p28 expression. Results are representative of 3 independent experiments, P values for all the panels using 1-way ANOVA with Bonferroni multiple comparison posttest (*P < .5, **P < .01, and ***P < .001).

Both interaction with PA28γ and PA28γ proteasomal activity are required for p30-mediated inhibition of tax/rex RNA. (A) p30 retains the ability to interact with exogenously expressed PA28γ in MEF PA28γ knockout cells. (B-C) p30-mediated repression of tax/rex RNA is reduced in MEF PA28γ−/− compared with MEF wild-type cells. p30 activity is rescued by exogenous expression of FLAG-PA28γ. (D) Characterization of FT-27 cells stably knocked-down for endogenous PA28γ. Western blots show drastically reduced PA28γ expression, whereas expression of related PA28α and PA28β were not affected by shRNA used. (E) p30 efficiently inhibits tax/rex mRNA in 293FT cells but not in FT-27 knocked down for PA28γ expression. Lack of p30-mediated repression was rescued by ectopic expression of a PA28γ expression vector. (F) In contrast to PA28γ, a PA28γ mutant defective for proteasome activation N151Y cannot rescue p30-mediated repression of tax/rex RNA. PA28γ (α21-26), unable to bind p30, was not able to rescue p30-mediated repression of tax/rex RNA. Western blots show p30 and PA28γ expression in transfected cells. (G) FLAG-PA28γ was transfected into 293T cells in the presence or absence of p30-HA or p28-HA expression vectors. Cell lysates were used for immunoprecipitations as indicated. (H) Repressive effects of p30 and p28 were analyzed by transfection of 293T cells with HTLV-1 molecular clone pBST, HTLV-1-LTR-luciferase reporter and either p30HA or p28HA as indicated. Western blots were performed to show relative levels of p30 and p28 expression. Results are representative of 3 independent experiments. (I) Repressive effects of p30 and p28 were analyzed by transfection of 293T cells with HTLV-2 molecular clone SV2neoHTLV-2, HTLV-1-LTR-luciferase reporter and either p30HA or p28HA as indicated. Western blots were performed to show relative levels of p30 and p28 expression. Results are representative of 3 independent experiments, P values for all the panels using 1-way ANOVA with Bonferroni multiple comparison posttest (*P < .5, **P < .01, and ***P < .001).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal