Dissection of the insulin signaling pathway via quantitative phosphoproteomics
- Marcus Krüger*,
- Irina Kratchmarova†,
- Blagoy Blagoev†,
- Yu-Hua Tseng‡,
- C. Ronald Kahn‡,§, and
- Matthias Mann*,§
- *Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany;
- †Department of Biochemistry and Molecular Biology, Center for Experimental BioInformatics, University of Southern Denmark, 5230 Odense, Denmark; and
- ‡Research Division, Joslin Diabetes Center and Harvard Medical School, Boston, MA 02215
-
Contributed by C. Ronald Kahn, December 21, 2007 (received for review November 27, 2007)
-
Fig. 1.Schematic overview of the differentiation protocol for immortalized brown adipocytes. (a) After SILAC labeling, cells were grown until confluence and differentiation was induced. At the indicated days, cells were stained with red oil, which is an indicator for differentiation. After 20 days, the differentiated cells were stimulated with insulin (5 min) for Western blot analysis. (b) After immunoprecipitation with the antiphosphotyrosine antibody (4G10) and SDS/PAGE, blots were probed with the same antibody. Diff. medium, differentiation medium; Ind., induction medium.
-
Fig. 2.Western blot analysis of selected insulin-dependent effectors. Brown adipocytes were stimulated with insulin for 5 min. After immunoprecipitation with an anti-phosphotyrosine antibody and SDS/PAGE, blots were probed with antibodies against the indicated proteins (Right). (Left) Represents the analysis of whole cell lysates to demonstrate equal loading. −, control, unstimulated cells; +, insulin treatment; IP, immunoprecipitation.
-
Fig. 3.Activation profiles of different categories of insulin-induced effectors. (a–h) To directly compare the kinetic profile of activated and deactivated effectors, all fold changes were normalized to zero (x − 1). Normalized inverted ratios were calculated for ratios smaller than one [1 − (1/x)]. (i) Western blot analysis of selected effectors. Brown adipocytes were stimulated for the indicated time intervals. After immunoprecipitation with anti-phosphotyrosine antibodies and SDS/PAGE, blots were probed with antibodies against the indicated proteins.
Footnotes
- §To whom correspondence may be addressed. E-mail: c.ronald.kahn{at}joslin.harvard.edu or mmann{at}biochem.mpg.de
- © 2008 by The National Academy of Sciences of the USA
