Fig. 4
T185 slightly enhanced FTD-linked CHMP2BIntron5-mediated endo/autosomal defects and neurotoxicity, compared to S185. a GFP (CTL), Flag-tagged T185, Flag-tagged S185, and/or CHMP2BIn5 were transfected into the HEK293T cells. Twenty-four to 48 h after transfection, the cells were incubated only with DMEM for 3 h and then treated with human epidermal growth factor (100 ng/mL) and cycloheximide (CHX, 30 μg/mL) for the indicated times (0, 30, 60, 120, or 240 min). Western blot analyses were conducted on the cell lysates with an anti-flag, anti-epidermal growth factor receptor (EGFR), anti-cMyc, or anti-GAPDH antibody. b The EGFR degradation was normalized to the levels of GAPDH. The graph represents the percentage of EGFR degradation that occurred during the indicated time. The percentage of EGFR degradation in each sample was normalized to that of EGFR in the control cells. The values are presented as the mean ± SEM of three independent replicates. One-way ANOVA followed by Tukey’s multiple-comparisons test; ***p < 0.001. c CHMP2BIn5-cMyc was cotransfected with or without flag-tagged T185 or flag-tagged S185 into HEK293T cells. Twenty-four to 48 h after transfection, the transfected cells or control cells were incubated with ammonium chloride (NH4Cl) or without it for 24 h, and then the cell lysates were subjected to western blot analyses with an anti-flag, anti-cMyc, anti-LC3, or anti-GAPDH antibody. d Autophagic flux indicates the difference in the LC3-II levels in the presence and in the absence of NH4Cl. The autophagic flux in the CHMP2BIn5-cMyc expressing cells with (or without) T185 or S185 expression was normalized to that of the control cells to represent autophagic ratio compared to the control cells. The values are presented as the mean ± SEM of three independent replicates. One-way ANOVA followed by Tukey’s multiple-comparisons test; ***p < 0.001. e Flag-tagged T185, flag-tagged S185 or/and CHMP2BIntron5 were cotransfected with GFP into cultured cortical neurons. The surviving neurons were counting the GFP-positive and propidium iodide (PI)-negative cells. The cell survival graph indicates the survival percentages of neurons expressing Flag-tagged T185 and/or CHMP2BIntron5-cMyc and GFP. The values are presented as the mean ± SEM of three independent replicates. One-way ANOVA followed by Tukey’s multiple-comparisons test; ***p < 0.001