Journal: Molecular Medicine Reports

Article Title: Aspirin inhibits glucose-6-phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites

doi: 10.3892/mmr.2016.5449

Figure Lengend Snippet: Aspirin-mediated acetylation of glucose-6-phosphate dehydrogenase (G6PD) is greater in (A) HCT 116 cells compared with in (B) HT-29 cells. Subconfluent cells were left untreated or were treated with aspirin for 24 h, lysates were prepared, and equal amounts of protein were immunoprecipitated with rabbit agarose-conjugated anti-acetyl lysine antibody. Agarose-bound proteins were eluted and immunoblotted with anti-G6PD antibody. (C) HCT-116 and (D) HT-29 samples were immunoblotted with anti-G6PD antibody. The experiments were repeated three times.

Article Snippet: The recombinant G6PD protein (isoform a ) was obtained from Origene Technologies, Inc. (Rockville, MD, USA).

Techniques: Immunoprecipitation

Journal: Molecular Medicine Reports

Article Title: Aspirin inhibits glucose-6-phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites

doi: 10.3892/mmr.2016.5449

Figure Lengend Snippet: Effects of aspirin on glucose-6-phosphate dehydrogenase (G6PD) activity in HCT 116 and HT-29 cells. Cells were cultured and left untreated or were treated with aspirin for 24 h. Protein (100 µ g) was used to conduct a G6PD assay. The reaction mixture was incubated at 37°C for 30 min, and absorbance was measured at 450 nm. G6PD activity was expressed as a percentage of control. The experiments were repeated three times. Data are represented as mean ± standard deviation. * P<0.05, ** P<0.01, *** P<0.001 vs. the control.

Article Snippet: The recombinant G6PD protein (isoform a ) was obtained from Origene Technologies, Inc. (Rockville, MD, USA).

Techniques: Activity Assay, Cell Culture, G6PD Assay, Incubation, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: Aspirin inhibits glucose-6-phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites

doi: 10.3892/mmr.2016.5449

Figure Lengend Snippet: Mass spectrometry (MS) analysis of recombinant glucose-6-phosphate dehydrogenase (G6PD) isoform a . In vitro acetylation of recombinant G6PD by aspirin. (A) A total of 5 ng in vitro acetylated recombinant G6PD was immunoblotted with anti-acetyl lysine antibody and the protein band was detected by enhanced chemiluminescence. (B-D) MS/MS fragmentation spectra showing acetyl modification of (B) K77, (C) K201 and (D) K235.

Article Snippet: The recombinant G6PD protein (isoform a ) was obtained from Origene Technologies, Inc. (Rockville, MD, USA).

Techniques: Mass Spectrometry, Recombinant, In Vitro, Tandem Mass Spectroscopy, Modification

Journal: Molecular Medicine Reports

Article Title: Aspirin inhibits glucose-6-phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites

doi: 10.3892/mmr.2016.5449

Figure Lengend Snippet: Location of aspirin-acetylated lysine residues identified in the present study and the naturally acetylated lysines on G6PD ( 25 ).

Article Snippet: The recombinant G6PD protein (isoform a ) was obtained from Origene Technologies, Inc. (Rockville, MD, USA).

Techniques:

Journal: Molecular Medicine Reports

Article Title: Aspirin inhibits glucose-6-phosphate dehydrogenase activity in HCT 116 cells through acetylation: Identification of aspirin-acetylated sites

doi: 10.3892/mmr.2016.5449

Figure Lengend Snippet: 3-Dimension space-filling model of recombinant glucose-6-phosphate dehydrogenase (G6PD; NP_000393), is shown. The location of aspirin-acetylated lysine residues are highlighted in blue (K77, K112, K119, K201, K235, K390, K396, K416, K438, K459, K462, K527, K538, K544).

Article Snippet: The recombinant G6PD protein (isoform a ) was obtained from Origene Technologies, Inc. (Rockville, MD, USA).

Techniques: Recombinant

Journal: International Journal of Molecular Sciences

Article Title: Insulin Resistance Does Not Impair Mechanical Overload-Stimulated Glucose Uptake, but Does Alter the Metabolic Fate of Glucose in Mouse Muscle

doi: 10.3390/ijms21134715

Figure Lengend Snippet: Insulin resistance does not impair overload-stimulated activation of the pentose phosphate pathway. After 12 weeks of high-fat diet (HFD) feeding, mice underwent unilateral synergist ablation surgery to induce overload (OVL) of the plantaris muscle for 5 days. ( A ) Muscles were excised and the protein content of glucose-6-phosphate dehydrogenase (G6PD) determined by immunoblot (IB) analysis. The G6PD antibody was validated using mouse tibialis anterior muscle samples overexpressing G6PDx (OE) or empty vector-transfected controls (-). ( B – D ) Ultra performance liquid chromatography/ multiple reaction monitoring-mass spectrometry (UPLC/MRM-MS) was used to measure 6-phosphogluconate, NADP and NADPH. ( E ) The ratio of NADP:NADPH was calculated. Data are mean ± standard deviation. Statistical analysis was performed using a 2-way ANOVA with Tukey’s post-hoc analysis. N = 7–9 muscles/group. p < 0.05 # vs. Sham (Sh); * vs. low-fat diet (LFD).

Article Snippet: Glucose-6-phosphate dehydrogenase (G6PD) and glutamine fructose-6-phosphate transaminase 1 (GFPT1) antibodies were validated using G6PDx or GFPT1 mouse muscle overexpression samples that were generated by transfecting the tibialis anterior muscles of male CD-1 mice (8 weeks old) with empty vector, untagged mouse G6PDx (cat#MC205271, OriGene Technologies, Rockville, MD, USA), or untagged mouse GFPT1 (cat#MC201036, OriGene Technologies, Rockville, MD, USA) vectors followed by standard in vivo muscle gene transfer/ electroporation procedures previously described by our lab [ , ].

Techniques: Activation Assay, Western Blot, Plasmid Preparation, Transfection, Liquid Chromatography, Mass Spectrometry, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Insulin Resistance Does Not Impair Mechanical Overload-Stimulated Glucose Uptake, but Does Alter the Metabolic Fate of Glucose in Mouse Muscle

doi: 10.3390/ijms21134715

Figure Lengend Snippet: Immunoblotting conditions.

Article Snippet: Glucose-6-phosphate dehydrogenase (G6PD) and glutamine fructose-6-phosphate transaminase 1 (GFPT1) antibodies were validated using G6PDx or GFPT1 mouse muscle overexpression samples that were generated by transfecting the tibialis anterior muscles of male CD-1 mice (8 weeks old) with empty vector, untagged mouse G6PDx (cat#MC205271, OriGene Technologies, Rockville, MD, USA), or untagged mouse GFPT1 (cat#MC201036, OriGene Technologies, Rockville, MD, USA) vectors followed by standard in vivo muscle gene transfer/ electroporation procedures previously described by our lab [ , ].

Techniques: Western Blot, Blocking Assay