Effects of RP depletion on nascent protein synthesis and activation of the IRBC. RPL7a and RPL11 shRNA vector-containing Eμ-Myc cell lines were treated for 22 hours with 1 μg/mL or 10 ng/mL doxycycline, respectively, to achieve ∼50% depletion of their corresponding RP mRNA. Renilla (Ren) shRNA-expressing cells, used as a control, were treated for 22 hours with 1 μg/mL doxycycline. (A) Rpl7a and Rpl11 transcript levels in correspondingly RP-depleted cells relative to control cells (shRen), determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and normalized to β-actin (n = 4). ****P < .0001 (unpaired Student t test). (B) Representative western blot analysis of RPL7a and RPL11 levels in whole cell lysates (WCLs) and in postribosomal supernatants (Post-ribo) containing 20 and 60 μg protein, respectively, from control (shRen) and from RPL7a- and RPL11-depleted cells (n = 2). (C) ³H-uridine autoradiogram (left panel) and ethidium bromide (EtBr)-stained agarose gel (right panel) of 28S and 18S shRNA from control (shRen) and from RPL7a- and RPL11-depleted cells pulsed with ³H-uridine for 2 hours. (D) Nascent protein synthesis rate, quantified by incorporation of ³H-leucine and normalized to total protein concentration (n = 3). *P < .05 (1-way analysis of variance (ANOVA) test). (E-F) Representative western blot analyses (E) WCLs (n > 4), post-ribosomal supernatants (Input), and coimmunoprecipitates of MDM2, p53, RPL11, and RPL5 in control (shRen) and RPL7a- and RPL11-depleted cells (n = 4) (F). A nonspecific rabbit immunoglobulin G (IgG) antibody was used as control for the immunoprecipitation (IP). a.u., arbitrary units; CASP3, caspase-3; cl., cleaved; cpm, counts per minute; long exp., long exposure. Data are presented as mean ± standard error of the mean (SEM).