Extended Data Fig. 4: Generation of Tmem206^−/− mouse lines and TMEM206 KD in HT-1080 cells.

(a) Strategy for disrupting Tmem206 in mice using CRISPR-Cas9 genome editing technique. gRNAs (in red) g1 and g5, both targeting exon 2, coding for the N terminus of TMEM206, were injected together and led to a 62 bp deletion. Similarly, g1 or g2, also targeting exon 2, were injected together with g6 targeting exon 3 after the sequence coding for the first transmembrane domain, leading to a deletion of ≈ 10 kbp. These injections led to the generation of three different Tmem206 KO mouse lines called T6-1/5 (injection of g1 and g5), T6-1/6 (injection of g1 and g6) and T6-2/6 (injection of g2 and g6). (b) Sequencing of one of the founders of the T6-1/5 line. (c) Genotyping PCR of WT homozygous (+/+), heterozygous (+/-) and KO homozygous (-/-) mice from the T6-1/5 line using the genotyping primers (a, in green) flanking the exon 2. (d) Western blot analysis of TMEM206 expression in different tissues from WT and KO mice confirmed that TMEM206 is deleted in all 3 different KO lines. Key experiments were done with BMDMs from these three different lines to exclude possible off-target effects of sgRNAs. 30 µg protein of membrane preparation were loaded per lane for each sample. TMEM206 proteins were detected using the custom-made antibodies targeted against the extracellular loop. Na/K-ATPase α1 subunit was used as loading control. For the WB done with animals from the T6-1/5 line, TMEM206 signals from different organs were obtained from different membranes but at the same exposure. This also holds true for the T6-1/6 line, except for the pancreas samples, which were obtained after shorter exposure time compared to the other organs. For the T6-2/6 line, all the signals come from the same membrane and exposure time. Na/K ATPase signals were obtained from different membranes and exposure time was different among the different organs (as organs express different amounts of Na/K ATPase) for the three lines. Lanes coming from different membrane or exposure time are delineated by dotted grey lines. Importantly, WT and KO samples (from the corresponding organs and mouse lines) always had the same exposure time to confirm the lack of TMEM206 expression in the KO animals. (e) TMEM206 knock-down (KD) efficiency in HT-1080 cells compared to cells transfected with non-targeting siRNA (ctrl), quantified in Fiji, 30 µg of protein per lane, n=5 (independent transfections). Na/K ATPase is a loading control. TMEM206 protein was detected using custom-made antibody against the extracellular loop. Mean ± s.e.m. Source numerical data and unprocessed blots are available in source data.

Source data