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. 2015 Feb 26;11(2):e1005002. doi: 10.1371/journal.pgen.1005002

Fig 8. MAB21L2 wild-type and mutant protein studies.

Fig 8

A. Western Blot analyses of MAB21L2 wild-type and mutant proteins (with N-terminal FLAG tag) in HLE-B3 cells. Please note the presence of wild-type MAB21L2 in both nuclear (N) and cytoplasmic (C) fractions (left), decreased expression of Arg51Gly and Arg51_Phe52del mutants (center) and the absence of Gln48* mutant protein (right; arrowhead indicates region corresponding to 5.5kDa, the predicted molecular weight for this peptide); predicted molecular weight for MAB21L2 is ~41kDa and for GAPDH is ~36 kDa; positions and corresponding molecular weights of protein ladder are shown for every blot. B. Cellular immunofluorescence analysis demonstrated no significant alteration in the localization pattern between wild-type and mutant (Arg51Gly and Arg51_52del) proteins with predominant nuclear localization and some cytoplasmic staining for all forms. C. RT-PCR analysis of MAB21L2 wild-type and mutant transcripts in human lens epithelial cells transfected with corresponding expression constructs. D. Protein stability assays with cycloheximide showed a more rapid decrease in the amount of Arg51Gly and Arg51_Phe52del mutant proteins in comparison to wild-type protein. Western blots signals were measured using ImageJ software, and obtained values were graphed to produce degradation curves represented by solid blue (wild-type), red (Arg51Gly) and green (Arg51_Phe52del) lines (standard deviations for every time point are indicated as thin vertical lines and exponential decay curves fitted into each graph are shown as dotted lines of corresponding colors); representative Western blot (WB) images are shown on the bottom (stability assays were performed in triplicate; 0, 1.5, 3, 4.5, 6, and 7.5 correspond to hours of exposure to cycloheximide). E. Summary of mRNA rescue experiments. Proportions of embryos with normal eyes in homozygous mab21l2Q48fs*5 embryos injected with wild-type human MAB21L2 mRNA, mRNA encoding the p.(Arg51Gly) mutant protein, and uninjected control larvae are shown; statistically significant (p<0.0005) differences are indicated with asterisks (***).