Extended Data Fig. 3: Effects of NCLX deletion on TMEM65 expression, validation of Nclx-3xFLAG mouse, and size exclusion chromatography standards.
From: TMEM65 regulates and is required for NCLX-dependent mitochondrial calcium efflux
a, Western blots for TMEM65 expression in Nclxfl/fl and Nclx−/− mouse embryonic fibroblasts, assessed 7 days after cre-mediated Nclx deletion. b, Densitometric quantification of western blots shown in (a). (n = 3 biological replicates/group; P-value by unpaired two-tailed t-test). Data are presented as mean ± SEM. c, Chromatogram of sequencing confirming proper in-frame insertion of DNA encoding the NCLX-3xFLAG epitope tag prior to the stop codon of the endogenous mouse Nclx gene. d, Genotyping gel showing pups heterozygous (+/−) or homozygous (+/+) for the 3xFLAG knock-in. Data are representative of >10 litters. e, Western blots validating solubilization and specific detection of NCLX-3xFLAG protein (arrow) in isolated adult mouse heart mitochondria. Amido black staining for total protein is shown as a loading control. f, Absorbance at 280 nm for gel filtration standards separated by size-exclusion chromatography on 1 MD column. g, Standard curve of elution fraction vs. known molecular weight for gel filtration standards separated by size-exclusion chromatography on 1 MD column. The equation shows the fit of the standards to a one-phase exponential decay. h, Absorbance at 280 nm for gel filtration standards separated by size-exclusion chromatography on 600 kD column. i, Standard curve of elution fraction vs. known molecular weight for gel filtration standards separated by size-exclusion chromatography on 600 kD column. The equation shows the fit of the standards to a one-phase exponential decay.
