Extended Data Figure 6: AxMLP morpholinos specifically and efficiently reduce AxMLP translation in cultured cells.

a, Schematic illustration of wild-type (WT) AxMlp (top) and N-terminal deletion AxMlp (bottom) constructs used to characterize AxMlp morpholino 1. The N-terminally deleted AxMlp lacks the morpholino-binding site. Both constructs have a His-tag on their C terminus (a). ED, effector domain; M, myristoylated N terminus domain; MH, MARCKS homology domain. b–i, Electroporated A1 myoblasts were stained with the indicated markers. b–e, Wild-type AxMlp plasmid was co-electroporated with the control morpholino (c) or the AxMlp-specific morpholino 1 (d), whereas wild-type AxMlp plasmid only (b) or wild-type AxMlp only without any primary antibody staining were used as negative controls (e). f–h, ΔN-AxMlp plasmid was co-electroporated with the control morpholino (g) or the AxMlp-specific morpholino 1 (h), whereas ΔN-AxMlp plasmid only (f) or pCMV-SPORT6-3C-His (empty vector) plasmid only served as negative controls (i). j, Western blotting for the cell lysates from the experiment above. AxMLP morpholino 1 specifically reduced AxMLP protein expression. k, Schematic illustration of the constructs used to characterize AxMlp morpholino 2. The original AxMlp expression clone from the cDNA library (BL212a101: top) was used as it included the 5′ untranslated region (UTR) target site for AxMlp morpholino 2. The subcloned AxMlp-His construct lacks the binding site for AxMlp morpholino 2 and was used as the control construct. l–r, Electroporated A1 myoblasts were stained with the indicated markers. l–n, BL212a101 plasmid was co-electroporated with the five-mismatch control morpholino (m) or the AxMLP-specific morpholino 2 (n), or BL212a101 plasmid only (l) or pCMV-SPORT6-3C-His (empty vector) plasmid only served as negative controls (o). AxMLP was detected using an anti-AxMLP antibody (red), and morpholinos were detected via FITC conjugation (green). p–r, AxMlp-3C-His plasmid was co-electroporated with the five-mismatch control morpholino (q) or the AxMlp-specific morpholino 2 (r) or AxMlp-3C-His plasmid only (p). AxMLP was detected using an anti-His-tag antibody (red), and morpholinos were detected via FITC conjugation (green). s, Western blotting for the cell lysates from the experiment above. AxMlp morpholino 2 specifically reduced AxMLP protein expression. Scale bars, 100 μm.