Fig. 6

β-TrCP mediating AMPK is involved in the ubiquitination degradation of PrlR. A–C Western blots of HC11 lysates for PrlR, n = 3. As shown in the left part of (A), the cells were treated with DMSO, Pitstop and EIPA to explore the effect of AMPK activation on the degradation of PrlR to explore the degradation mode of PrlR. In the right part of (A), similar methods have also been used to explore the organelles involved in the degradation of PrlR. (B) and (C) are the quantifications of protein expression in the above two experiments, respectively. (Pitstop: pinocytosis inhibitor, EIPA: endocytosis inhibitor, MG132: proteasome inhibitor, MA: lysosomal inhibitor) D–G Western blots of HC11 lysates for AMPK/β-TrCP/PrlR pathway, n = 3. In (D), bands were incubated with indicated proteins and Actin antibodies, n = 3. Protein expressions of β-TrCP, PrlR and milk proteins are shown in (E), (F) and (G), respectively, n = 3. H Immunoblot analysis of PrlR immunoprecipitates in HC11, probed for AMPK/Ub-PrlR pathway. After being cultured in the designated medium (all containing MA), cells were lysed by CHAPS lysis buffer, and lysate were not only used for immunoprecipitation to detect the ubiquitination of PrlR but also for WB to detect AMPK/PrlR pathway. I Immunoblot analysis of PrlR immunoprecipitates in HC11, probed AMPK/β-TrCP/Ub-PrlR pathway. After being transinfected by siRNA and cultured in the designated medium (all containing MA), cells were lysed by CHAPS lysis buffer, and lysate were not only used for immunoprecipitation to detect the ubiquitination of PrlR but also for WB to detect AMPK/PrlR pathway. In this figure, cells for 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