Fig. 6
CPEB2 binds to and promotes PDGFRα mRNA translation. a Western blot analysis of PDGFRα protein levels in P3 and P10 lungs from CP2WT and CP2KO mice (n = 3 mice per group). b RNA immunoprecipitation (RNA-IP). P10 lung lysates were precipitated with control (Ctrl) or CPEB2 (CP2) IgG. RT-qPCR of PDGFRα and PDGF-A mRNA levels in immunoprecipitates expressed as relative ratio to the non-target control GAPDH. Data are mean ± s.e.m. from 3 independent experiments. c Dual luciferase reporter assay. Three CPEs and hexanucleotide (Hex) in the 3′-UTR of mouse PDGFRα mRNA are marked. The reporter plasmids, one of FLuc constructs and RLuc, were co-transfected with the plasmid expressing myc-tag or myc-CPEB2 (myc-CP2) into HeLa cells. Analysis of FLuc and RLuc activity and the expression level of myc-CP2. Data are mean ± s.e.m. from 4 independent experiments. d Primary MYF cultures from P10 lungs were harvested at 1, 2 and 3 days in vitro (DIV) for immunoblotting. e Similar to d, except DIV3 MYF cultures were harvested for RT-qPCR of PDGFRα mRNA level relative to GAPDH. Data are mean ± s.e.m. from 4 independent cultures. f Similar to d, except DIV1 CP2WT and CP2KO MYFs were infected with lentiviruses expressing GFP or myc-CP2 and harvested at DIV3 for immunoblotting. Het: Cpeb2+/− MYFs. d,f Protein lysates were from 2 independent cultures. g PDGF-A–induced Ki67 expression in serum-starved MYFs. Lentivirus-infected CP2WT and CP2KO MYFs were starved in 0.5% serum for 24 h and then treated with PDGF-A for the indicated time, followed by labeling of DAPI, Ki67 and αSMA. Data are mean ± s.e.m. from 3 independent experiments. n.s. not significant, *P < 0.05, **P < 0.01 and ***P < 0.001, by Student’s t test in b, one-way ANOVA in c and two-way ANOVA in g