α345 (IV) trimer assay of clinical α5(IV) mutants with N-terminal tag. (a,b) Luminescence was measured in the cells (a) or media (b) in HEK293T cells transiently expressing α3, α4, and N-terminal-tagged α5(IV) WT or the indicated mutant. Error bars are the mean ± SD (n = 4). ∗P < 0.01, ∗∗P < 0.001 versus WT (Tukey-Kramer test). (c) Scatterplot of the % relative light units (RLU) of intracellular (a)/secreted (b) from cells expressing WT or mutant α5 chain. Solid line: Y = X. (d) Intracellular residual rate calculated from the measured value of RLU of intracellular/(intracellular + secreted) from cells expressing WT or mutant α5(IV) chain. Error bars indicate the mean ± SD (n = 4). ∗P < 0.01, ∗∗P < 0.001 versus WT (Tukey-Kramer test). Black, WT; blue, possibly of single-nucleotide polymorphisms; gray, α5(IV) mutants with mild pathology; red, α5(IV) mutants with severe pathology.