Figure 7

Detection of MCLK1 in protein fractions. Western blot analysis of nuclear protein extracts prepared from mouse embryonic fibroblasts (MEFs) (a) and Western blot analysis of nuclear protein fractions from mouse tissues (b). Mclk1 KO cells were generated as previously published³⁴ and the KO tissue samples were obtained from agoMclk1 KO mice described in ref. 47. They were used to further confirm band identity. The wild-type controls are Mclk1 floxed MEFs or tissues without Cre expression. In both panels, 40 µg proteins per lane were loaded except for the membrane fraction (MF), where 5 µg were used. HDAC1 was used as a loading control of nuclear soluble fractions (NF). For insoluble (chromatin-bound) nuclear fractions (CBF), lamin A/C and Histone H3 served as markers. The mitochondrial matrix protein SOD2 was used as an additional control to validate the purity of the nuclear protein fractions. (a) In MEFs, MCLK1 could only be detected in membrane fractions (MF). (b) In mouse tissues, MCKL1 could be detected in both nuclear soluble fractions (NF) and membrane fractions (MF). However, the mitochondrial matrix protein SOD2 could also be detected in both fractions suggesting that the nuclear soluble fraction was contaminated by mitochondria. MCLK1 could not be detected in the chromatin-bound nuclear fraction (CBF). No evidence therefore points to the presence of MCLK1 in the nucleus or an association with chromatin. Uncropped western blot scans with size indications are shown in Supplementary Data.