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. 2016 Nov 28;36(24):3019–3032. doi: 10.1128/MCB.00303-16

FIG 3.

FIG 3

PDCD2L associates with late 40S precursors. (A) GFP-PDCD2L SILAC copurification results plotted by SILAC ratios on the x axis (intensity of peptides originating from the GFP-PDCD2L purification versus the control purification), which reflects signal specificity, and signal intensity on the y axis (total peptide intensity for each protein normalized to molecular mass), which reflects the relative abundance of each protein in the purification. Proteins presenting SILAC ratios of >2.0 and <2.0 are shown. Factors known to be associated with late pre-40S particles are shown in orange. (B) Western analysis of total extracts (lanes 1 and 2) and Flag immunoprecipitates (lanes 3 and 4) prepared from HEK 293 cells that stably express Flag-tagged versions of PABPN1 (lanes 1 and 3) and PDCD2L (lanes 2 and 4). (C) Western analysis of endogenous proteins using fractions of centrifuged sucrose gradients that were prepared by using extracts of HEK 293 cells. The positions of the 40S, 60S, and 80S sedimentations are indicated at the bottom. (D) Schematic of 18S rRNA biogenesis in human cells. Following cleavage at sites 01/A′, A0, 1, and 2, the 21S pre-rRNA is matured into the 18S-E precursor in the nucleus. Final maturation of the 18S-E pre-rRNA into mature 18S rRNA occurs in the cytoplasm. Probes used to detect the 18S-E pre-rRNA (red) as well as mature 18S (blue) and 28S (green) rRNAs are indicated. ETS, external transcribed spacer; ITS, internal transcribed spacer. (E) Western blot (WB) (top two panels) and Northern blot (NB) (bottom three panels) analyses of protein and RNA, respectively, prepared from total cell extracts (lanes 1 and 2) and Flag immunoprecipitates (lanes 3 and 4) using HEK 293 cells that stably express Flag-tagged versions of PABPN1 (lanes 1 and 3) and PDCD2L (lanes 2 and 4). The 18S-E, 18S, and 28S RNAs were detected by using the specific probes shown in panel D. (F) Quantification of 18S-E pre-rRNA enrichment. 18S-E levels were normalized to levels of mature 18S rRNA. The values were then set to 1.0 for the control Flag-PABPN1 purification. Data and error bars represent the averages and standard deviations, respectively, from three independent experiments.