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. Author manuscript; available in PMC: 2019 Jun 19.
Published in final edited form as: Oncogene. 2018 Dec 19;38(16):2937–2952. doi: 10.1038/s41388-018-0639-8

Figure 5: Analysis of PTBP1 and NOVA1 binding to DR8 using RNA baits.

Figure 5:

(A) Cartoon schematic displaying hTERT mRNA. A large polypyrimidine tract located upstream of DR8 was included in our analysis. Five oligo baits (oligo 1-5) were designed to encompass ~ 1 kb of hTERT mRNA. Baits were then in vitro transcribed into RNA and biotinylated in order to tether on to streptavidin beads. (B) Whole cell lysates from H1299 and Calu6 were used to test PTBP1 binding to the 5 baits. Eluted samples from the RNA-IP were probed via western blot for PTBP1. (C) and (D) Oligo 4 was mutated by altering all T residues to A. PTBP1 was then RNA-IP and compared with wild type (WT) bait 4. Western blot probing of PTBP1 showed a reduction in PTBP1 binding to the mutant bait. Quantification of western blot band intensity determined a significant reduction in PTBP1 binding (n = 3, Student’s t test set at p < 0.05 for significance, *). (E) and (F) Mutant bait was designed for oligo 3 converting all YCAY NOVA1 binding motifs to YAAY. NOVA1 in the elution was probed via western blot and quantified. (n = 3, Student’s t test set at p < 0.05 for significance, *).