Fig. 7

Loss of PGC-1α facilitates milk fat production due to its acetylation regulated by AMPK. A and B TAG concentration in cell (A) and medium (B), n = 5. In the experiment, cells (transfected by siRNA in advance) for TAG determination were collected after 24 h incubation with 0 mM glucose medium, 10 mM glucose medium and (10 mM glucose + 300 μM Phe) medium. C Western blots of HC11 lysates for AMPK/PGC-1α signaling pathway, n = 3. D and E Western blots of HC11 lysates for AMPK/p38/PGC-1α signaling pathway, n = 3. Cells were treated with 10 mM glucose medium and (10 mM glucose + 300 μM Phe) medium in the case of DMSO and SB203580 (p38 inhibitor) for 24 h, respectively. In (D), bands were incubated with target proteins and Actin antibodies, n = 3. Protein expressions of target proteins are shown in (E), n = 3. F and G Western blots of HC11 lysates for AMPK/Sirt1/PGC-1α signaling pathway, n = 3. Cells were treated with 10 mM glucose medium and (10 mM glucose + 300 μM Phe) medium in the case of control-knockdown and Sirt1-knockdown for 24 h, respectively. In (F), bands were incubated with target proteins and Actin antibodies, n = 3. Protein expressions of target proteins are shown in (G), n = 3. H Immunoblot analysis of PGC-1α immunoprecipitates in HC11, probed for acetylation of PGC-1α. Cells were lysed by CHAPS lysis buffer, and lysate were not only used for immunoprecipitation to detect the acetylation of PGC-1α but also for WB to detect AMPK/PGC-1α pathway. In this figure, cells for TAG determination and WB were collected after 24 h incubation with indicated medium. All datas with error bars are averages ± SEM. In histograms, the numbers above the column are the p-values marked as a range if significantly different (P < 0.05) and each small black dot represents a test value