Fig. 6.

ARPP21 and miR-128 have antagonistic functions on an overlapping set of target mRNAs. a, b Conserved TargetScan predictions for miR-128-binding sites (orange bars) and ARPP21 iCLIP CL signals (turquoise) at the 3′UTRs of the miR-128 target mRNAs Phf6 and Msk1. c Schematic of 3′UTR GFP-reporter assay to assess direct effects of miR-128 and ARPP21 on individual transcripts. d, e Phf6 and Msk1 3′UTR reporter fluorescence upon miR-128 and/or ARPP21 expression normalized to control transfection. miR-128 represses and ARPP21 increases GFP reporter expression; co-transfection leads to intermediate expression. Data expressed as mean ± s.d., ***p < 0.001, Student’s t-test, n = 3. f Representative immunoblot analysis of endogenous PHF6 and MSK1 protein levels in 293T cells. ARPP21 transfection leads to increased protein expression of MSK1 and PHF6. miR-128 mimic transfection results in reduced expression of MSK1 and PHF6 protein. Co-transfection leads to intermediate levels. g, h Quantification of PHF6 and MSK1 protein expression from five independent experiments as in f. *p < 0.05, ***p < 0.001, one-sample t-test against 100%, data represents mean ± s.d. i miR-128 overexpression by lentivirus in primary cortical neurons at DIV7 reduces MSK1 and PHF6 protein expression. The arrowhead marks the specific PHF6 band. j Quantification of miR-128 overexpression effect on MSK1 and PHF6 protein levels. One-sample t-test against 100%, n = 4 biological replicates, *p < 0.05; **p < 0.01. Data expressed as mean ± s.d. Protein levels of ARPP21, MSK1, and PHF6 upon ARPP21 overexpression (k) and knockdown (l) in primary cortical neurons at DIV7. Quantification of protein expression upon overexpression (m) or knockdown (n) of ARPP21. One-sample t-test against 100%, n = 6 biological replicates, *p < 0.05. Data expressed as mean ± s.d. Full western blot images are presented in Supplementary Fig. 21