Fig. 5: FBXW7 binds and degrades the N-terminal MYRF.
a Schematic showing workflow of primary rat OPC isolation, expansion, and electroporation with either a dominant-negative Fbxw7 construct (CMV-3xFLAG-Fbxw7ΔFBox), pooled siRNAs against Fbxw7 or non-targeting controls (siControl). b LC-MS peptide counts for significant proteins enriched by anti-FLAG pull-down in 3xFlag-Fbxw7ΔFBox electroporated cells normalized to GFP electroporated controls. Mean ± SEM, (N = 4 independent cell isolations and IPs). Statistical significance determined by multiple unpaired, two-tailed Student’s t test. c IP-western blot for MYRF following pull down of FBXW7∆FBox from cultured rat OLs at 72 h differentiation. Representative blot shown from 3 independent experiments. d Western blot analysis of the N-MYRF cleavage product in siFbxw7 or siCont electroporated cells at 24 or 48 h differentiation. Western blots were repeated 7 times with 5 biological replicates. e Western blot of MYRF in siRNA electroporated OLs treated with cycloheximide (CHX) for 4 h. Relative intensity of the N-MYRF band quantified in f. Mean ± SEM, N = 4 independent cell isolations. Statistical significance determined by unpaired, two-tailed Student’s t test. g Representative images of MBP and MAG expression in cultured OLs differentiated for 48 or 72 h after electroporation with siControl or siFbxw7. h, i MBP+ and MAG+ OLs normalized to total OLIG2+ cells. Mean ± SEM, N = 6 independent cell isolations with 2 technical replicates (coverslips) per isolation. Statistical significance determined by two-way ANOVA. j qRT-PCR for myelin genes on siRNA treated cells. Mean ± SEM, N = 3 (independent cell isolations) with 2 technical replicates (qRT-PCR). Statistical significance determined by multiple unpaired, two-tailed Student’s t test. k Quantification of EdU incorporation in primary rat OPCs following siControl or siFbxw7 electroporation. Mean ± SEM, N = 3 independent cell isolations with 4 technical replicates (coverslips). Statistical significance determined by unpaired, two-tailed Student’s t test. l–n Proteins with +/- > 1.2 fold change were sorted by gene ontology (GO) terms “lipid metabolism,” “myelin,” and “adhesion”. N = 4 independent cell isolations with 2 technical replicates. o Table of the top 10 enriched proteins by TMT-LS/MS in siFbxw7 electroporated OLs relative to siControl electroporated OLs at 3 days differentiation. Proteins with 1 or more MYRF ChIP-Seq peaks within 50 kb of the transcription start site of their corresponding gene identified based on previously published data50. Created in BioRender. Emery, B. (2024) BioRender.com/w60g354.
