Figure 2

Analysis of Pomt1 mRNA and protein expression, and α-dystroglycan glycosylation. (a) PCR analysis of Pomt1 alleles in DNA extracted from mouse retinas: Pomt1 floxed, 1,475 bp; Pomt1 wild-type, 1,392 bp; Pomt1 null, 545 bp. Genotypes defined by the results are indicated above each lane, abbreviated as indicated below. Last lane, DNA ladder. (b) Results of RT-PCR analysis designed to detect Pomt1 transcripts. PCR products from the wild-type (182 bp) and null (136 bp) transcripts were detected in Pomt1^lox/lox and Pomt1 cKO mice, respectively. Gapdh mRNA (238 bp) levels showed no differences across the different mouse retinas. (c) qRT-PCR analysis of RNA extracted from the retinas of Pomt1^lox/lox, Pomt1^+/− and Pomt1 cKO mice. The left graph shows the levels detected (mean ± SD, n = 4) of the Pomt1 wild-type transcript, and the right graph the Pomt1 null transcript levels (mean ± SD, n = 4). (Statistical significances: *p = 0.0299; **p = 0.0058; ***p < 0.001). (d) Expression of POMT1 in the mouse retina. Immunoreactivity of POMT1 in the inner segments of photoreceptors (green) of control mice (Pomt1^lox/lox in the picture) was virtually lost in the retina of Pomt1 cKO mice. Nuclei were stained with DAPI (blue). Abbreviations: IS, inner segments; ONL, outer nuclear layer. (e) Western blotting analysis of α-DG glycosylation (VIA4-1, upper panel), laminin-binding ability (middle panel) and β-DG levels (lower panel) in WGA-enriched retinal extracts. Uncropped DNA gels and blot images are presented in Supplementary Figs S6a,b and S7. Genotype abbreviations: Pomt1^+/lox refers to the Pomt1^+/lox/Crx-Cre⁻ genotype; Pomt1^lox/lox refers to Pomt1^lox/lox/Crx-Cre⁻; Pomt1^+/− refers to Pomt1^+/lox/Crx-Cre⁺; and Pomt1 cKO refers to Pomt1^lox/lox/Crx-Cre⁺.