Fig. 2: MUC1 interacts with ATAD3A through the CQC motif, which is necessary for mitophagy. | Cell Death & Disease

Fig. 2: MUC1 interacts with ATAD3A through the CQC motif, which is necessary for mitophagy.

From: The oncoprotein MUC1 facilitates breast cancer progression by promoting Pink1-dependent mitophagy via ATAD3A destabilization

Fig. 2

A Proteins of HeLa229 cells precipitated by anti-MUC1-C or IgG antibody were analyzed by mass spectrometry and co-IP. The proteins from the co-IP assay were separated by SDS-PAGE and stained with Coomassie brilliant blue. Screening of proteins localized in mitochondria, including ATAD3A. B–D Co-IP assay to detect the interaction of endogenous MUC1 and ATAD3A. Anti-MUC1-C immunoprecipitates from MDA-MB-468 (B), BT549 (C) and HeLa229 (D) cells were analyzed by western blotting with the indicated antibodies (left). Anti-ATAD3A immunoprecipitates from MDA-MB-468 (B), BT549 (C), and HeLa229 (D) cells were analyzed by western blotting with the indicated antibodies (right). E The schematic shows GFP fused with different truncations of MUC1-CD (upper). HEK293T cells were transfected with GFP-MUC1-CD truncated mutant plasmids, subjected to a co-IP assay with an anti-ATAD3A antibody, and then analyzed by western blotting using anti-GFP and anti-ATAD3A antibodies. F Co-IP assay of MUC1-CD and ATAD3A in HEK293T cells. HEK293T cells were cotransfected with ATAD3A and pEGFP-MUC1-CD or pEGFP-MUC1-CD-AQA. Cell lysates were subjected to denatured co-IP. G Purified recombinant GST, GST-MUC1-CD, or GST-MUC1-AQA was incubated with ATAD3A. The precipitated beads were detected by western blot using anti-ATAD3A and anti-GST antibodies. H Co-IP of endogenous MUC1 and ATAD3A in HeLa229 cells treated with 30 μM CCCP (2 h) or 30 μM Mdivi-1 (2 h). After IP with IgG or anti-ATAD3A, the immunoprecipitates were analyzed with anti-MUC1-C and anti-ATAD3A antibodies. I HeLa229/g-MUC1 cells were transfected with vector, MUC1-CD-WT and MUC1-CD-AQA plasmids and then treated with DMSO, 30 μM CCCP (24 h) or 30 μM Mdivi-1 (24 h). Western blotting was performed using antibodies as indicated. Quantification of mitochondrial proteins was calculated. The data represent the mean ± SD from three independent experiments. Differences between linked groups were evaluated using a two-tailed Student’s t test. *P < 0.05, **P < 0.01; ***P < 0.001; NS not significant.

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