Figure 3.

The Premature-Stop-Codon Mutation in LNPK Results in Loss of Transcript and Full-Length Protein

(A) Graph showing the expression of human LNPK in relation to the geometric mean of three housekeeping genes via quantitative RT-PCR. Compared are control fibroblasts (control) and fibroblasts derived from the unaffected mother (B-II-2(M)) and the affected girl (B-III-2(A)) from family B. Consistent with NMD due to a premature stop codon, LNPK levels are reduced by approximately 50% in B-II-2(M) and almost completely lost in B-III-2(A). n = 3 independent experiments.

(B) Western blot images probing for full-length LNP and β-actin in parallel blots with the same input. Shown are triplicates for the three conditions described before. Neither full-length nor truncated LNP signal could be detected for B-III-2(A).

(C) Quantification of blots shown in (B). LNP was normalized to β-actin. For B-III-2(A), no peaks could be identified for quantification. Note that the severe transcript reduction is not reflected at the protein level for B-II-2(M). Shown are individual data points, as well as the mean ± SEM.

(D–F) Images show representative examples of fibroblasts stained with DAPI and the antibody detecting LNP for all three conditions. Results confirm the absence of full-length protein in cells derived from the affected girl. The scale bar in (D) represents 10 μm.