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. Author manuscript; available in PMC: 2021 Aug 17.
Published in final edited form as: Hum Mutat. 2021 May 24;42(7):877–890. doi: 10.1002/humu.24218

Figure 2. Functional analyses of MAB21L1 and variant proteins.

Figure 2.

A. Western Blot. Western blot analysis of N-terminal FLAG tagged MAB21L1 wild-type and Arg51Leu, Arg62Cys and Gly220Arg variants. Constructs were expressed in HLE-B3 cells. β-actin was used as a loading control. The proteins correspond to their expected molecular weight (~41kDa MAB21L1 and ~42kDa β-actin). B. ELISA. N-terminal FLAG tagged MAB21L1 wild-type and Arg51Leu, Arg62Cys, and Gly220Arg variants were transfected into HLE-B3 cells. Cell lysates were assessed for FLAG-tagged protein expression; protein levels of Arg51Leu and Arg62Cys were found to be significantly affected. C. Immunocytochemistry. N-terminal FLAG tagged MAB21L1 wild-type and variants were transfected into HLE-B3 cells and stained for FLAG (green) and DAPI (blue; cell nuclei). Wild-type and variant proteins can be found within the cell nucleus, indicating no disruption in localization. D. In vivo complementation assays. Proportion of phenotypically normal embryos at 24-hpf in progeny of heterozygous mab21l2Q48Sfs*5 crosses injected with wild-type or variant Arg51Leu, Arg62Cys or Gly220Arg MAB21L1 mRNA. Statistical significance is indicated by asterisks; * P≤0.05 ** P≤0.01 *** P≤0.001; error bars indicate SEM. UN: uninjected; 1-MAB21L1-WT; 2-MAB21L1-Arg51Leu; 3-MAB21L1-Arg62Cys; 4-MAB21L1-Gly220Arg.