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. 2018 Nov 6;115(47):E11061–E11070. doi: 10.1073/pnas.1809609115

Fig. 3.

Fig. 3.

PTBP1 inhibits miR-124-1 maturation in vivo. (A) Mature miR-124 is enhanced in a Ptbp1 KO cell line. The levels of pri-miRNAs were measured in WT and Ptbp1 KO mESCs by RT-PCR (Left). PCR in the absence of reverse transcription (−RT) controlled for genomic DNA contamination. Spanning primer pairs as diagrammed in SI Appendix, Fig. S2B were used for RT-PCR analysis. A representative gel image is shown from three biological replicate cultures (Left). The levels of mature miR-124, miR-294, and miR-9 were measured in WT and Ptbp1 KO mESCs by RT-qPCR (Right), normalized to U6 snRNA, and further normalized to the first replicate of WT cells (n = 3 cultures; Student’s t test; **P ≤ 0.01; error bars are SEM). Ptbp1 depletion in the KO line was confirmed by Western blot using a PTB_CT antibody targeting the common C-terminal peptide of PTBP1 and PTBP2 (Bottom Right) (50). PTBP1 and PTBP2 isoforms are indicated to the left. Alpha-tubulin served as loading control. Representative gel images from three cultures. (B) Ectopic PTBP1 reverses enhancement of miR-124 expression in the KO cell line. Mature miR-124, miR-294, and miR-9 were measured by RT-qPCR in WT and Ptbp1 KO mESCs after ectopic FLAG-PTBP1.4 expression (Top). MiRNA levels were normalized to U6 snRNA, and further normalized to the first replicate of WT cells with no transfected PTBP1 (n = 3 biological replicates; Student’s t test; ***P ≤ 0.001; error bars are SEM). Expression of FLAG-PTBP1.4 was confirmed by Western blot. GAPDH served as a loading control (Bottom). Representative gel images are from three replicates. (C) Reintroduction of PTBP1 inhibits miR-124 expression in cultured cortical neurons. Cortical neurons were transduced with FLAG-PTBP1.4 expressing AAV 1 d after plating, and mature miRNA levels were measured at day 8 by RT-qPCR. Mir-9 and let-7e were assayed as controls (Top). Mature-miRNA level was normalized to U6 snRNA, and further normalized to the tRFP-only condition in each replicate (n = 3 biological replicates; Student’s t test; **P ≤ 0.01; error bars are SEM). Expression of FLAG-PTBP1.4 and endogenous PTBP2 were confirmed by Western blot. GAPDH served a loading control (Bottom). Representative gel images from three replicates. n.s, not significant.