Figure 4.

Immunoblots and Flow Cytometry Confirm that α-DG Glycosylation Is Reduced in Muscle and Fibroblasts of Affected Individuals with GMPPB Mutations

(A) Immunoblot analysis of skeletal-muscle protein lysates from P1 and P4. (Ai) The membrane was incubated with IIH6 and β-DG antibodies. P1 shows a reduction in α-DG glycosylation. (Aii) For P4, WGA-enriched skeletal-muscle homogenates were used, and the immunoblot was probed with IIH6, α-DG core, and β-DG antibodies, as well as by laminin overlay. β-DG appears as a possible doublet in P1 and P4 (and P8, Figure S6).

(B) Immunoblot analysis of fibroblast protein lysate from P1, P2, P4, P5, and P6. The membrane was incubated with IIH6 and β-DG antibodies. α-DG glycosylation is reduced in fibroblasts of individuals with GMPPB mutations.

(C) Flow cytometry of fibroblasts revealed reduced α-DG glycosylation for P1, P2, P4, P5, P6, and P7. This histogram shows the MFI of IIH6 staining for a secondary-only control, P1, P2, P4, P5, P6, P7, and a control fibroblast cell line. The MFI of the populations positive for IIH6 were 88.95 for the control, 19.6 for P1, 30.15 for P2, 26.34 for P4, 14.60 for P5, 28.63 for P6, and 45.3 for P7 (n = 6 for all). There was a statistically significant (p < 0.05) reduction in α-DG glycosylation in fibroblasts from the six individuals tested compared to controls, as determined by the average MFI of IIH6. The percentage of fibroblasts (out of a minimum of 10,000) that were positive for the IIH6 epitope was 85% for the control, 27% for P1, 16% for P2, 45% for P4, 60% for P5, 50% for P6, and 65% for P7. A IIH6-positive gate was set up with a negative control (where primary antibody was omitted) for all fibroblast cell lines tested and the control.