Fig. 5: MaTAR25 interacts with PURB to carry out its function.

a Scatterplot depicts the fold enrichment of protein candidates from isobaric tags for the relative and absolute quantitation (iTRAQ) analysis comparing two independent oligo pair sets targeting MaTAR25 RNA transcripts vs Ppib RNA transcripts. b Upper: immunoblot analysis of PURA and PURB following pulldown of MaTAR25 or Ppib from 4T1 cells. Lower: immunoblot analysis of PURB following the pulldown of MaTAR25 or Ppib from 4T1 cells or 4T1 MaTAR25 KO cells. More than three different experiments were performed, and representative images are shown. c MaTAR25, Ppib, and Gapdh transcripts were assessed by qRT-PCR in endogenous PURB, or IgG (negative control) immunoprecipitates from 4T1 cells. Fold enrichment of PURB associated RNA signal over IgG signal is calculated and data are presented as mean values ± SD (n = 3 independent experiments). *P < 0.05 (paired Student’s t test; two-tailed). Immunoblot analysis of PURB was performed as a control. d qRT-PCR analysis and immunoblotting of Tns1 expression in 4T1 cells following ectopic overexpression of PURB. The relative expression levels are shown as mean values ± SD (n = 2 independent experiments). e ChIP-qPCR analysis of PURB occupancy over the identified MaTAR25 targeting region and non-targeting region of the Tns1 DNA locus by ChIRP-seq analysis. ChIP-qPCR was performed in 4T1 control1 cells, 4T1 MaTAR25 KO1 cells, and upon ectopic expression of MaTAR25 in MaTAR25 KO1 cells. Primers for a MaTAR25 non-targeting region and the Gapdh TSS were used as negative controls. Bar graphs represent the mean ± SD (n = 2 independent experiments).