Figure 6.
N-domain dimer interface residues influence CRT multimerization. (A, C, and E) Lysates from HEK293T cells expressing indicated untagged full-length CRTWT or CRTDel52 constructs or control transfected cells (Vec) were separated by native-PAGE (8% gels; top left panels) or SDS-PAGE under reducing (8% gels; bottom left panels) or nonreducing (4–20% gradient gels; right panels) conditions and immunoblotted with the indicated antibodies. (B, D, and F) Left panels: WT or mutant CRT monomer bands from native-PAGE immunoblots in panels A, C, and E were normalized relative to the corresponding total CRT signal from the reducing SDS-PAGE immunoblots. Data show mean ± SD; the normalized signals were log-transformed, and the statistical significance was assessed via one-way repeated measures ANOVA (D and F) or one-sample t test (B). Right panels: Quantification of CRT monomer/monomer + multimer (total) bands from the nonreducing SDS-PAGE immunoblots. Data show mean ± SD, and the statistical significance was assessed via one-way repeated measures ANOVA (D and F) or one-sample t test (B). Data were averaged over five independent blots from four to five independent transfections (B and D) or two independent blots from two independent transfections (F), each with two to three protein loads. MW, molecular weight.