Figure 2

Functional analyses of MAB21L1 and variant proteins. (a) Western blot analysis. 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) Enzyme‐linked immunosorbent assay. 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 4ʹ,6‐diamidino‐2‐phenylindole, dihydrochloride (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 the progeny of heterozygous mab21l2^Q48Sfs*5 crosses injected with wild‐type or variant Arg51Leu, Arg62Cys, or Gly220Arg MAB21L1 messenger RNA. UN, uninjected; hpf, hours postfertilization; 1‐MAB21L1‐WT; 2‐MAB21L1‐Arg51Leu; 3‐MAB21L1‐Arg62Cys; 4‐MAB21L1‐Gly220Arg. Statistical significance is indicated by asterisks; *p ≤ .05, **p ≤ .01, and ***p ≤ .001; error bars indicate SEM