Fig. 1

Development and validation of TMEM106B overexpression model. A Design of the transgene and induction of transgenic expression using Cre-mediated recombination. B Western blot of hemibrain lysates of 6-month-old animals using a human-specific anti-TMEM106B antibody (60,333–1-Ig, Proteintech), eGFP antibody (50430–2-AP, Proteintech), and anti-TMEM106B (E7H7Z, Cell Signaling Technology) (n = 3/genotype). C Quantification of the Western blot shows expression of the transgene, with a 2-fold increase in homozygous animals relative to heterozygous animals. D Quantification of total TMEM106B protein levels shows an approximately 4-fold to 8-fold overexpression for heterozygous and homozygous animals, respectively. E qPCR analysis of hemibrain lysates of 6-month-old animals using mouse-specific and human-specific qPCR primers shows expression of the transgene without downregulation of mouse Tmem106b (n = 4/genotype). F phase-contrast images of MEFs showing large vacuolar structures and G quantification of the vacuolar phenotype using pixel-based analysis. Per genotype, 4 replicate wells (derived from one embryo) were used and phase contrast images from 8 positions per well were acquired. Datapoints represent the average vacuolization ratio per image. Data represented as mean ± SEM. One-way ANOVA **, P < 0,01; ***, P < 0,001; ****, P < 0,0001