Skip to main content
. 2007 Feb 8;26(4):1081–1093. doi: 10.1038/sj.emboj.7601563

Figure 5.

Figure 5

Phosphorylation of the TTS site is metabolically regulated. (A) Protein extracts from approximately 10 mg liver, 20 mg lung and 200 mg epididymal fat pad isolated from fed and fasted WT and fed TTS mice were analyzed by Anderson PAGE and Western blotting. Western blots were developed using anti C/EBPα antibody (14AA). The phosphorylated and non-phosphorylated C/EBPα p42 and p30 forms are indicated. * indicates a nonspecific cross-reactive band. (B) NIH3T3 cells were infected with virus encoding wild-type C/EBPα or mutant C/EBPα T222A,T226A. Cells were analyzed for C/EBPα expression and Thr 222,226 phosphorylation by Western blotting using polyclonal anti-C/EBPα antibody (14AA) or monoclonal anti-C/EBPα phospho-Thr 222,226 (2C6), respectively. (C) Nuclear lysates prepared from 2 mg liver or 40 mg epididymal fat pads from fed or fasted WT mice were analyzed for C/EBPα content and threonine 222, 226 phosphorylation as in (B). Only the C/EBPα p42 form is shown for simplicity. Western blotting was normalized using anti-MAPK p42/44 antibody (MAPK). (D) Liver protein lysates was prepared from WT and TTS mice during a glucose tolerance test (Figure 7E). Mice were killed at the indicated time points and analyzed as in (A). Protein lysate from approximately 10 mg liver was loaded in each lane. (E) Nuclear lysates prepared from 2 mg liver from WT mice during a glucose tolerance test (as in D) were analyzed for C/EBPα content and T222,T226 phosphorylation by Western blotting as in (C).