FIG 8.

Human PDCD2 and PDCD2L proteins contribute to 40S ribosomal subunit production. (A) Western blot analysis showing depletion of PDCD2 from wild-type HeLa cells (lanes 1 and 2) as well as PDCD2L-null HeLa cells (1A3) (lanes 3 and 4). (B) Sucrose gradient analysis of total extracts prepared from wild-type (a and b) and PDCD2L-null (c and d) HeLa cells that were previously treated with PDCD2-specific (b and d) or control nontarget (a and c) siRNAs. The positions of free small (40S) and large (60S) ribosomal subunits, monosomes (80S), and polysomes are indicated. (C) Northern blot analysis of mature and precursor rRNAs using total RNA prepared from WT (lanes 1 and 2) and PDCD2L-null (1A3) (lanes 3 and 4) cells that were previously treated with PDCD2-specific (lanes 2 and 4) or control nontarget (lanes 1 and 3) siRNAs. Pre-rRNAs were detected by using a probe complementary to sequences in the 5′ ITS region. Pre-rRNA species are indicated on the left. (D and E) 18S-E (D) and 21S (E) pre-rRNA levels were normalized to the 28S rRNA level and are expressed relative to values for cells treated with control siRNAs. Data and error bars represent the means and standard deviations, respectively, from six independent experiments. **, P value of <0.01, as determined by Student's t test. (F) GFP-PDCD2 SILAC copurification results plotted as SILAC ratios on the x axis and signal intensities on the y axis. Proteins presenting SILAC ratios of >2.0 and <2.0 are indicated. GFP-PDCD2, RPS2, and PRMT3 are shown in color.