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. 2017 Sep 5;8(43):73516–73528. doi: 10.18632/oncotarget.20640

Figure 2. Pabpn1 regulates ATG expression through altered PAS utilization and nuclear export, affecting translation and protein abundance.

Figure 2

Experiments were performed in stable scrambled control shRNA (scram) or Pabpn1-shRNA (shPab) transfected C2C12 mouse myoblast cultures under normal growth conditions. A. Bar chart shows mRNA fold change of ATG. Fold change was calculated after normalization to Hprt housekeeping gene and control cultures. Ct values were obtained with the proximal primer set. B. Bar chart shows the ratio between products from distal primer and proximal primer set for five ATGs. C. Western blot analysis of protein lysate from cytoplasmic and nuclear fractions. Gapdh marks the cytoplasmic fraction and LaminA the nuclear fraction. D. Bar charts show the abundance of transcripts from distal primer set or proximal primer set between nuclear and cytosolic fractions in scram or shPab cell cultures. Hprt is shown as an unchanged reference gene. E. Bar chart shows the enrichment of transcripts from the distal primer set in the ribosomal bound fraction in scram or shPab cell cultures. Hprt is shown as an unchanged reference. F. Image shows a representative Western blot of Pabpn1, Atg3, Lc3, and Wipi1 from scram or shPab C2C12 cell cultures. Gapdh is used as loading control. G. Bar chart shows the ratio of protein accumulation (Atg5, Lc3a and Wipi1) between scram and shPab cell cultures. Protein abundance was normalized to Gapdh loading control. H. Western blot analysis of PABPN1 immunoprecipitation (IP), which was used to isolate RNA from PABPN1 complex. IP was carried out with antibodies to PABPN1 and control was carried out with beads only. Immunoblot was carried out with anti-PABPN1 antibodies and tubulin as loading control. I. Bar charts show the RNA-IP enrichment of transcripts from distal primer set or proximal primer set in scram or shPab cell cultures. RIP enrichment was calculated from input and Hprt. Averages and standard deviations are from three biological replicates. Student’s T-Test was used to assess statistical significance (0.05<p <0.01 (*); 0.01<p<0.005 (**) P <0.005 (***).