Fig. 4. PTBP1 bound exon 30 and adjacent intronic areas of ITSN1 RNA and modulated splicing of ITSN1.

A In vitro RNA pull-down assay in LN229 cell line. Proteins were subjected to western blot analysis with anti-PTBP1 antibody after pull-down. B Analysis of PTBP1 CLIP-seq data from GSE19323 and GSE114720, the blue lane shows PTBP1 CLIP peaks for exon 30 and adjacent area of introns of ITSN1. C The primer position used to detect the binding segment of PTBP1 and ITSN1 pre-mRNA in the RIP experiment. D PTBP1 protein precipitation upon RIP with anti-PTBP1. E ITSN1 amplification from the anti-PTBP1 coprecipitated RNA fraction. F RIP with anti-PTBP1 followed by RT–qPCR quantification, values were expressed as mean ± SD from three independent experiments (two-tailed Student’s t test, **P < 0.01, ***P < 0.001). G The potential binding sequences of PTBP1 within ITSN1 E30 RNA used in the RNA pulldown assay and its position in the ITSN1 pre-mRNA, the red font represented the mutated base. H RNA pulldown assay was used to detect the binding of PTBP1 to the biotinylated ITSN1 E30 WT RNA and ITSN1 mutant RNA. I Schematic representation of the ITSN1 minigene and the primer sets for PCR detection of ITSN1 exon 30-included and -excluded products. The primers for the internal reference of exogenous ITSN1-minigene were CMV-F and E29-R. The mutated base sequence of the ITSN1-minigene was marked with red font. J Western blot analysis for the inhibition of PTBP1 in transient cells transfected with the wild-type and mutant ITSN1-minigene plasmids. K Representative pictures of agarose PCR gels depicting the inversion in the splicing pattern of ITSN1 regulated by PTBP1 after transfected with the wild-type and mutant ITSN1-minigene plasmids. L RT–qPCR analysis showing changes in the ratio of ITSN1-S/ITSN1-L upon silencing PTBP1 in LN229 cells expressing ITSN1 minigene or ITSN1 mutant minigene. Values were expressed as mean ± SD from three independent experiments (two-tailed Student’s t test, ***P < 0.001).