Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease
Article Title: PFKFB2 Is Pivotal for Metabolic Flexibility and Differential Glucose Utilization
doi: 10.1161/JAHA.125.043921
Figure Lengend Snippet: A and B , Overall protein O‐GlcNAcylation was measured by western blot, using heart homogenate from fed and fasted, control (CON) and PFKFB2 knockout (cKO) hearts (n=7–9). O‐GlcNAc levels on western blot were measured using the average of signals from 2 different pan‐specific antibodies targeting the modification (a mouse monoclonal antibody (RL2 clone) and a mixed rabbit monoclonal antibody), and quantified either as the full lane of proteins ( A ) or only proteins that are 50 kDa or larger ( B ). C , GFAT1 was measured by western blot, using heart homogenate from fed and fasted, control and cKO hearts (n=7–9). D and E , O‐GlcNAcylation of Sp1 was measured by immunoprecipitation of Sp1, subsequently blotting for both Sp1 and O‐GlcNAc (RL2 antibody), and taking the quotient (n=5). Sp1 was quantified directly ( E ), and the ratio of RL2 signal/Sp1 signal was used to determine the relative O‐GlcNAcylation of Sp1 ( D ). F and G , OGT ( F ) and OGA (MGEA5; G ) were measured by western blot, using heart homogenate from fed and fasted, control and cKO hearts (n=7–9). Data were analyzed by 2‐way ANOVA with Šídák’s post hoc test for multiple comparisons between groups that differed by 1 factor. * P ≤0.05, *** P ≤0.001, **** P ≤0.0001. Data are shown as mean ± SD. cKO indicates PFKFB2 knockout; CON, control; GFAT1, glutamine‐fructose 6‐phosphate amidotransferase 1; OGA, O‐GlcNAcase; O‐GlcNAc, O‐linked N‐acetylglucosamine; OGT, O‐GlcNAc transferase; Sp1, specificity protein 1; and UDP‐GlcNAc, uridine diphosphate N‐acetylglucosamine.
Article Snippet: GFAT1 , , CST: 5322S , 1:500 , 1:2000.
Techniques: Western Blot, Control, Knock-Out, Modification, Immunoprecipitation
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