Fig 2: Effect of PKCe on MARCKS phosphorylation in the ARC in glucose-challenged mice and in GT1-7 cells exposed to glucose. (a) AgRP-PKCe KO mice and Flox-PKCe controls were challenged with glucose (1g/kg ip) and brains fixed by cardiac perfusion with saline/formaldehyde after 30 min. Cryosections were subjected to immunofluorescence using antibodies against phospho-MARCKS. 3V, 3rd ventricle. Sections shown are typical of 4 biological replicates per group. Intensity of phospho-MARCKS staining was quantified within the defined areas of interest that corresponded to the area of Cre expression and PKCe depeletion in Fig. 1b; Student’s t-test, ∗P< 0.05. (b) GT1-7 cells were infected with adenovirus mediating the co-expression of GFP and kinase-dead (KD) or constitutively active (CA) PKCe as indicated. Cells were exposed to the concentrations of glucose indicated, or with 5 mM glucose as well as 5 mM 2-deoxyglucose (2DG), for 4 hours. Cell extracts were subjected to SDS-PAGE and immunoblotting with the antibodies indicated. Representative immunoblots of three experiments carried out in triplicate are shown. (c) The results of densitometry (means ± SEM) of AMPKa phosphorylation. Two-way ANOVA: P<0.01, main effect of glucose. (d) The results of densitometry (means ± SEM) of MARCKS phosphorylation. Two-way ANOVA: P<0.001, main effect of PKCe. One-way ANOVA: ∗ P<0.05, ∗∗ P<0.01 vs GFP.