Journal: Molecular Metabolism
Article Title: Skeletal muscle O-GlcNAc transferase is important for muscle energy homeostasis and whole-body insulin sensitivity
Figure Lengend Snippet: OGT knockout results in augmented Interleukin-15 production in skeletal muscle. (A) Expression of myokines in the gastrocnemius muscles of WT and mKO mice fed HFD for 22 wks (B) IL-15 mRNA expression in skeletal muscles from clamped mice (n = 8–10, 18-week-old male mice). (C) Serum IL-15 levels in WT and mKO mice fed HFD for 22 wks (n = 10). (D) Oil red-O staining of cultured stromal vascular fraction cells isolated from mouse IngW and induced to differentiate in the presence or absence of gastrocnemius muscle extracts from WT (top row) and mKO (bottom row) mice with (right) or without (left) IL15 neutralizing antibodies. Scale bar, 1 mm. (E) Expression of lipogenic genes in IngW (n = 10). (F) Immunoprecipitation was performed using anti-EZH2 and anti-O-GlcNAc antibodies respectively, and immunoblotted with O-GlcNAc and EZH2 antibodies respectively. (G – I) Chromatin immunoprecipitation using antibodies against O-GlcNAc (G), EZH2 (H), and H3K27me3 (I), respectively. Quantitative RT-PCR was used to quantify IL-15 promoter sequence as detailed in Methods. *p
Article Snippet: Antibodies used for Western blot analyses were: anti-OGT (#SAB2101676 Sigma), anti-O-GlcNAc (#2739 Abcam), anti-ATP synthase β subunit (#A-21351 ThermoFisher Scientific), anti-succinate dehydrogenase A subunit (SDHA) (#14715 Abcam), OGT (#5368 CST), O-GlcNAc (CTD110.6 #9875 CST), OGA (#124807 Abcam), Hexokinase II (#2867 CST), PFK1 (#166722 Santa Cruz Biotechnology (SCBT)), PDH (#C54G1 CST), PDH pS293 (#ABS204 Millipore), PDH pS300 (#ABS194 Millipore), ACC pS79/257 (#07–300 Millipore), Insulin receptor β (#3025 CST), IRS1 (#23822 CST), AKT 1/2/3 (#9272 CST), AKT pT308 (#9275 CST), AKT pS473 (#9271 CST), AKT1 (#5298 SCBT), TBC1D4 (#07–741 Millipore), TBC1D4 pS711 (Custom), TBC1D4 pS324 (Custom), ATGL (#2138 CST), HSL (#4107 CST), HSL pS563 (#4139 CST), GFAT1 (#125069 Abcam), GLUT4 (#PA1-1065 Thermo Fisher).
Techniques: Knock-Out, Expressing, Mouse Assay, Staining, Cell Culture, Isolation, Immunoprecipitation, Chromatin Immunoprecipitation, Quantitative RT-PCR, Sequencing