Premium
Characterization of ATP10C and multiple signaling pathway proteins in C2C12 cells using Immunoblot Assays and Immunofluorescence Microscopy
Author(s) -
Hurst Sarah Elizabeth,
Minkin Steven,
Biggerstaff John,
Dhar Madhu
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.530.7
Subject(s) - immunofluorescence , c2c12 , microbiology and biotechnology , indirect immunofluorescence , characterization (materials science) , chemistry , biology , antibody , myocyte , immunology , nanotechnology , materials science , myogenesis
Atp10c is a putative phospholipid translocase which encodes for a type IV P‐type ATPase and is a strong candidate for diet‐induced obesity and diabetes in both mice and humans. To characterize the ATP10C protein, and to identify its molecular targets, immunofluroescence and immunoblot assays were carried out. Using an ATP10C‐GFP construct, the ATP10C protein was estimated to be about 165kD. The ATP10C‐GFP fusion protein was found to co‐localize with a commercially available anti‐human ATP10C. ATP10C appears to be localized to the nuclear membrane along with a punctate pattern in and around the nucleus, suggesting an endosomal/trans‐golgi organization, strengthening its biological role in protein trafficking in exocytic and endocytic pathways. Furthermore, when Atp10c expression was silenced (>70%) using transient transfection with ATP10C‐specific siRNA sequences, glucose uptake decreased (2.5 fold) and resulted in a significant up‐regulation of p38 and p44/42 (p‐value<0.05) proteins. Comparing the mean fluorescence intensity per cell between wild type and Atp10c‐silenced C2C12 myotubes revealed that there was a significant (p‐value<0.05) down regulation in Akt2 (>6 fold) and a significant up‐regulation of GLUT1 (>25 fold) and IRS‐1 (>19 fold). Concluding from these results, ATP10C appears to be a newly identified protein, affecting glucose uptake via either and/or both MAPK and PI3K signaling. Research support for this project was provided by the Center of Excellence, College of Veterinary Medicine, University of Tennessee, Knoxville, and the American Diabetes Association.