Fig. 6: Phosphorylation is required for CPEB1 to regulate translation.

a pCPEB1 immunostaining of QSCs, fiSCs and 4 hours cultured SCs (12 hours after mice were sacrificed). Nuclei were stained with DAPI. (n = 3 independent experiments). b The WT or CPE mutated 3′UTR of Myod1 was inserted into the pmiR-report luciferase vector and co-transfected with pcDNA3.0-flag (Control), CPEB1, or CPEB1 (T171A, S177A) mutant into 293 T cells along with the Renilla vector as an internal control. 36 hours after transfection, luciferase activity was quantified by the dual-luciferase assay. (n = 3 independent experiments). c, d FACS-isolated SCs were infected with the indicated adenovirus for 36 hours. c Myod1 immunostaining on SCs infected with adenovirus-containing GFP, CPEB1, or CPEB1 (T171A, S177A). d Quantification of Myod1 RFUs on SCs after the indicated virus infection. (n = 3 independent experiments, the number of quantified cells is 166, 197, and 182 for the GFP, CPEB1, and CPEB1 (T171A, S177A) groups, respectively. e C2 cells were transfected with CPEB1-mVenus or CPEB1 (T171A, S177A)-mVenus. After 48 hours, C2 cells were harvested and used for mVenus RNA-IP. Myod1 mRNA level was analyzed by qRT-PCR. (n = 3 independent experiments). f–h Proteomic analysis of CPEB1 interacting proteins. f Schematic illustration of the workflow for CPEB1-associated protein analysis. g The number of spectra of CPEB1 proteins detected in C2 cells transfected with CPEB1-mVenus or h CPEB1 (T171A, S177A)-mVenus after immunoprecipitation. (n = 2 independent experiments). i Venn diagram of CPEB1 and phosphorylation mutated CPEB1-specific interacting proteins. j Functional enrichment analysis of corresponding proteins subsets in i. Data are presented as mean ± SD in panels b, d, e. The p-values calculated by two-tailed unpaired t-test are used for comparing two groups in b, d, and e, ns not significant. Source data are provided as a Source Data file.