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Fig. 2 | Molecular Brain

Fig. 2

From: TMEM106B, a frontotemporal lobar dementia (FTLD) modifier, associates with FTD-3-linked CHMP2B, a complex of ESCRT-III

Fig. 2

The T185 variant of TMEM106B was more associated with endogenous CHMP2B and reduced autophagic flux compared to the S185 variant. a Flag-tagged T185 or Flag-S185 was transfected into the HEK293T cells. Twenty-four to 48 h after transfection, a protein coimmunoprecipitation assay was performed with an anti-flag antibody. Western blot analyses were performed with an anti-CHMP2B or anti-flag antibody. b The bar graph represents the percentage of interaction of T185 or S185 with endogenous CHMP2B. For quantification, the band intensity of CHMP2B from the immunoprecipitation samples was normalized to that of either flag-T185 or flag-S185. The values are presented as the mean ± SEM of three independent replicates. Student’s t-test, **p < 0.01 c Each flag-tagged TMEM106B SNP (T185 or S185) was transfected into cultured cortical neurons. Twenty-four hours after transfection, the neurons were fixed and stained with an anti-flag antibody and anti-TMEM106B antibody. The arrows indicate the colocalization of CHMP2B with either T185 or S185. Scale bar: 20 μm. d The bar graph indicates the colocalization of endogenous CHMP2B with either T185 or S185 of TMEM106B. The coefficients of colocalization were measured and analyzed with the ImageJ program (Colocalization Indices). The values are presented as the mean ± SEM of three independent replicates. Student’s t-test; **p < 0.001. e Flag-tagged T185 or Flag-tagged S185 was transfected into HEK293T cells. Twenty-four h after transfection, the transfected or control cells were incubated with or without ammonium chloride (NH4Cl) for 24 h. The cell lysates were then subjected to western blot analyses with an anti-flag, anti-microtubule-associated protein 1A/1B-light chain 3 (LC3), or anti-GAPDH antibody. f The autophagic flux indicates the difference in the LC3-II levels in the presence and absence of NH4Cl. The autophagic flux ratio in the T185- or S185-expressing cells was normalized to that of 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 < 0.01, ***p < 0.001

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