Journal: Biochemical Journal

Article Title: Histone deacetylase 7 activates 6-phosphogluconate dehydrogenase via an enzyme-independent mechanism that involves the N-terminal protein-protein interaction domain

doi: 10.1042/BCJ20240380

Figure Lengend Snippet: HEK293 cells were transfected with an empty vector (EV) control or expression constructs encoding 6PGD (Flag-tagged) and HDAC7 wild type (WT) or HDAC7 enzyme-dead (ED) (V5-tagged) at a molar ratio of 1:1 (6PGD:HDAC), after which the enzymatic activity of 6PGD, class IIa HDAC activity and protein expression in cell lysates was measured. ( A ) Kinetics of 6PGD enzymatic activity. Cell lysates were incubated with 6PGD substrate at 37°C for 60 min, with 6PGD enzyme activity being measured every 5 min. Results shown are from one representative experiment of three independent biological repeats. ( B ) 6PGD enzymatic activity in HEK293 cells. Absorbance at two timepoints from the linear phase of the kinetic curves ( A ) was used to calculate ΔOD, which was then applied to the standard curve generated using NADPH to determine 6PGD activity (nmol/min/ml). Data (mean ± SEM) are combined from three independent experiments and are displayed as relative activity (normalised to levels in cells transfected with 6PGD alone). Statistical analysis was performed using repeated measure one-way ANOVA followed by Dunnett's multiple comparison (* P < 0.05; ** P < 0.01). ( C ) Class IIa HDAC enzymatic activity in transfected cells. Cell lysates were incubated with the class IIa HDAC fluorophore-conjugated substrate BOC-Lys(trifluoroacetyl)-AMC for 30–60 min at 37°C, after which stop reagent was added and fluorescence measured. Data (mean ± range) are combined from two independent experiments. ( D ) Protein expression of 6PGD (anti-Flag), HDAC7 WT and HDAC7 ED (anti-V5) in transfected HEK293 cells was assessed by immunoblotting, with GAPDH used as a loading control. Data shown are representative blots, with similar results apparent in three independent experiments.

Article Snippet: Membranes were then probed with anti-Flag (mouse anti-Flag, 4 μg/ml, Sigma-Aldrich, F3165; rabbit anti-Flag, 0.8 μg/ml, Sigma-Aldrich, F7425; rabbit anti-Flag, 0.414 μg/ml, Cell Signaling Technology, 14793) or anti-V5 antibodies (mouse anti-V5, 1 μg/ml Bio-Rad, MCA1360; rabbit anti-V5, 0.192 μg/ml, Cell Signaling Technology, 13202) at 4°C overnight.

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Construct, Activity Assay, Incubation, Generated, Comparison, Fluorescence, Western Blot

Journal: Biochemical Journal

Article Title: Histone deacetylase 7 activates 6-phosphogluconate dehydrogenase via an enzyme-independent mechanism that involves the N-terminal protein-protein interaction domain

doi: 10.1042/BCJ20240380

Figure Lengend Snippet: HEK293 cells were transfected with 6PGD-Flag and HDAC7-V5, HDAC7 ED-V5 or HDAC6-V5 expression constructs at a molar ratio of 1:1 (6PGD:HDAC) for 24 h. Cells were then lysed and cell lysates were immunoprecipitated using an anti-V5 antibody ( A ) or an anti-Flag antibody ( B ), after which the interaction between 6PGD and HDAC7/HDAC6 was assessed by immunoblotting using antibodies against Flag and V5. Tubulin was also probed as a loading control. The displayed immunoblots are representative of two experiments ( A ) or are from a single experiment ( B ). The red arrow indicates a shorter form of 6PGD (6PGD-s) that was detected by anti-Flag immunoblotting.

Article Snippet: Membranes were then probed with anti-Flag (mouse anti-Flag, 4 μg/ml, Sigma-Aldrich, F3165; rabbit anti-Flag, 0.8 μg/ml, Sigma-Aldrich, F7425; rabbit anti-Flag, 0.414 μg/ml, Cell Signaling Technology, 14793) or anti-V5 antibodies (mouse anti-V5, 1 μg/ml Bio-Rad, MCA1360; rabbit anti-V5, 0.192 μg/ml, Cell Signaling Technology, 13202) at 4°C overnight.

Techniques: Transfection, Expressing, Construct, Immunoprecipitation, Western Blot, Control

Journal: Biochemical Journal

Article Title: Histone deacetylase 7 activates 6-phosphogluconate dehydrogenase via an enzyme-independent mechanism that involves the N-terminal protein-protein interaction domain

doi: 10.1042/BCJ20240380

Figure Lengend Snippet: ( A ) Schematic diagram of HDAC7 constructs used for studying the interaction between HDAC7 and 6PGD. HDAC7-s, spliced, full-length HDAC7; HDAC7-u, unspliced isoform lacking the first 22 amino acids (NM_001204278.1); N-HDAC7, N-terminal domain of HDAC7; C-HDAC7, C-terminal domain of HDAC7. ( B ) Immunoprecipitation of 6PGD (anti-Flag). HEK293 cells were transfected with 6PGD-Flag and N-HDAC7-V5, C-HDAC7-V5 or SOX7-V5 (unrelated control) expression constructs at a molar ratio of 1:1 (6PGD:HDAC or SOX7) for 24 h. Cells were then lysed and cell lysates were immunoprecipitated using an anti-Flag antibody, after which the interaction between 6PGD and HDAC7 proteins was assessed by immunoblotting using antibodies against V5. GAPDH levels were assessed as a loading control. The displayed immunoblots are from one experiment that is representative of two independent experiments. The red arrow indicates a shorter form of 6PGD (6PGD-s) that was detected by anti-Flag immunoblotting.

Article Snippet: Membranes were then probed with anti-Flag (mouse anti-Flag, 4 μg/ml, Sigma-Aldrich, F3165; rabbit anti-Flag, 0.8 μg/ml, Sigma-Aldrich, F7425; rabbit anti-Flag, 0.414 μg/ml, Cell Signaling Technology, 14793) or anti-V5 antibodies (mouse anti-V5, 1 μg/ml Bio-Rad, MCA1360; rabbit anti-V5, 0.192 μg/ml, Cell Signaling Technology, 13202) at 4°C overnight.

Techniques: Construct, Immunoprecipitation, Transfection, Control, Expressing, Western Blot

Journal: Biochemical Journal

Article Title: Histone deacetylase 7 activates 6-phosphogluconate dehydrogenase via an enzyme-independent mechanism that involves the N-terminal protein-protein interaction domain

doi: 10.1042/BCJ20240380

Figure Lengend Snippet: HEK293 cells were transfected with expression constructs encoding 6PGD (Flag-tagged) and full-length, N-HDAC7 or C-HDAC7 (V5-tagged) at a molar ratio of 1:1 (6PGD:HDAC) for 24 h, after which the enzymatic activity of 6PGD, class IIa HDAC activity and protein expression in cell lysates was measured. ( A ) Kinetics of 6PGD enzymatic activity. Cell lysates were incubated with the 6PGD substrate 6-phosphogluconate at 37°C for 60 min, with 6PGD enzyme activity being measured every 5 min. Results shown are from one representative experiment of three independent biological repeats. ( B ) 6PGD enzymatic activity in HEK293 cells. Absorbance at two timepoints from the linear phase of the kinetic curves ( A ) was used to calculate ΔOD, which was then applied to the standard curve generated using NADPH to determine 6PGD activity (nmol/min/ml). 6PGD activity was normalised to total protein concentration in cell lysates and data (mean ± SEM) are combined from three independent experiments, displayed as relative activity (normalised to levels in cells transfected with 6PGD alone). ( C ) Class IIa HDAC activity in cells transfected with the indicated constructs. Cell lysates were incubated with the class IIa HDAC-specific fluorophore-conjugated substrate BOC-Lys(trifluoroacetyl)-AMC for 30–60 min at 37°C, after which stop reagent was added and fluorescence measured. Data (mean ± SEM) are combined from three independent experiments (normalised to levels in cells transfected with EV). ( D ) Protein expression of 6PGD (anti-Flag) and HDAC7 (anti-V5) in transfected cells was assessed by immunoblotting, with GAPDH used as a loading control. Samples were analysed on two different SDS-PAGE gels to distinguish 6PGD and the truncated forms of HDAC7. The displayed immunoblots are representative of three independent experiments. The red arrow indicates a shorter form of 6PGD (6PGD-s) that was detected by anti-Flag immunoblotting. Statistical analyses ( B , C ) were performed using repeated measure one-way ANOVA followed by Dunnett's multiple comparison (ns, not significant; * P < 0.05; *** P < 0.001).

Article Snippet: Membranes were then probed with anti-Flag (mouse anti-Flag, 4 μg/ml, Sigma-Aldrich, F3165; rabbit anti-Flag, 0.8 μg/ml, Sigma-Aldrich, F7425; rabbit anti-Flag, 0.414 μg/ml, Cell Signaling Technology, 14793) or anti-V5 antibodies (mouse anti-V5, 1 μg/ml Bio-Rad, MCA1360; rabbit anti-V5, 0.192 μg/ml, Cell Signaling Technology, 13202) at 4°C overnight.

Techniques: Transfection, Expressing, Construct, Activity Assay, Incubation, Generated, Protein Concentration, Fluorescence, Western Blot, Control, SDS Page, Comparison

Journal: Biochemical Journal

Article Title: Histone deacetylase 7 activates 6-phosphogluconate dehydrogenase via an enzyme-independent mechanism that involves the N-terminal protein-protein interaction domain

doi: 10.1042/BCJ20240380

Figure Lengend Snippet: HEK293 cells were transfected with expression constructs encoding 6PGD (Flag-tagged) and full-length HDAC7, N-HDAC7, C-HDAC7 or SOX7 as an unrelated control protein (all V5-tagged) at a molar ratio of 1:1 (6PGD:HDAC7 or SOX7) for 24 h. ( A ) Cell lysates were immunoblotted (left) or were incubated with disuccinimidyl suberate to enable crosslinking of proteins, then immunoblotted (right). After the reaction was quenched by the addition of Tris-HCl, the same amounts of cell lysates from each transfection were used for immunoblotting to examine protein expression (left), as well as 6PGD dimerization and/or higher order complex formation (right). The levels of transfected proteins, including 6PGD monomers and dimers, were assessed by immunoblotting with antibodies against V5 and Flag, with GAPDH used as a loading control. Samples were analysed on the same SDS-PAGE gel, and fluorescent secondary antibodies were used to distinguish monomeric 6PGD and truncated HDAC7. The displayed immunoblots are from one experiment, representative of two independent experiments using different crosslinkers (similar results were obtained using glutaraldehyde as the crosslinker). ( B ) Quantification of the two higher order 6PGD-containing complexes across different transfection conditions. Data (displayed relative to the 6PGD alone control) are combined from two independent experiments and show the combined levels of the 150 and 200 kDa complexes.

Article Snippet: Membranes were then probed with anti-Flag (mouse anti-Flag, 4 μg/ml, Sigma-Aldrich, F3165; rabbit anti-Flag, 0.8 μg/ml, Sigma-Aldrich, F7425; rabbit anti-Flag, 0.414 μg/ml, Cell Signaling Technology, 14793) or anti-V5 antibodies (mouse anti-V5, 1 μg/ml Bio-Rad, MCA1360; rabbit anti-V5, 0.192 μg/ml, Cell Signaling Technology, 13202) at 4°C overnight.

Techniques: Transfection, Expressing, Construct, Control, Incubation, Western Blot, SDS Page

Journal: Biochemical Journal

Article Title: Histone deacetylase 7 activates 6-phosphogluconate dehydrogenase via an enzyme-independent mechanism that involves the N-terminal protein-protein interaction domain

doi: 10.1042/BCJ20240380

Figure Lengend Snippet: HEK293 cells were transfected with expression constructs encoding 6PGD (Flag-tagged), full-length HDAC7, N-HDAC7 and/or C-HDAC7 (V5-tagged) at a molar ratio of 1:1 (6PGD:HDAC) for 24 h. ( A ) Cells were then lysed, and the same amounts of protein lysates were used as input (top) and an affinity assay (bottom) using Cibacron Blue-conjugated agarose beads (100–200 mesh). Input lysates and agarose-precipitated proteins were then subjected to immunoblotting (anti-Flag, anti-V5) to assess levels of transfected proteins and the binding of 6PGD to Cibacron Blue. GAPDH was used as loading control. The displayed blots are from one experiment, representative of two independent experiments. The red arrow indicates the shorter form of 6PGD (6PGD-s). ( B ) Quantitative analysis of 6PGD levels relative to GAPDH. Total 6PGD levels were determined by quantifying the intensity of both 6PGD-s and full-length 6PGD in the input and Cibacron Blue pulldown samples, relative to respective GAPDH expression levels on immunoblots. Data are mean ± range from two independent experiments. ( C ) Quantitative analysis of 6PGD-s levels. Relative levels of 6PGD-s were quantified by comparing to the total combined band intensity of both 6PGD-s and full-length 6PGD on immunoblots of input lysates of HEK293 cells transfected with 6PGD and HDAC7, N-HDAC7 or C-HDAC7 from data presented in , , and . Data (mean ± SEM) are combined from 5 to 7 independent transfection experiments. Statistical analysis was performed using Kruskal–Wallis test followed by Dunn's multiple comparison (ns, not significant; ** P < 0.01; *** P < 0.001).

Article Snippet: Membranes were then probed with anti-Flag (mouse anti-Flag, 4 μg/ml, Sigma-Aldrich, F3165; rabbit anti-Flag, 0.8 μg/ml, Sigma-Aldrich, F7425; rabbit anti-Flag, 0.414 μg/ml, Cell Signaling Technology, 14793) or anti-V5 antibodies (mouse anti-V5, 1 μg/ml Bio-Rad, MCA1360; rabbit anti-V5, 0.192 μg/ml, Cell Signaling Technology, 13202) at 4°C overnight.

Techniques: Transfection, Expressing, Construct, Western Blot, Binding Assay, Control, Comparison

Journal: Biochemical Journal

Article Title: Histone deacetylase 7 activates 6-phosphogluconate dehydrogenase via an enzyme-independent mechanism that involves the N-terminal protein-protein interaction domain

doi: 10.1042/BCJ20240380

Figure Lengend Snippet: HEK293 cells were transfected with expression constructs encoding 6PGD (Flag-tagged) and class IIa HDACs (HDAC4, HDAC5, HDAC7 or HDAC9, V5-tagged) at a molar ratio of 1:1 (6PGD:HDAC), after which the enzymatic activity and levels of 6PGD in cell lysates was measured. ( A ) 6PGD enzymatic activity in HEK293 cells. Data (mean ± SEM) are combined from four independent experiments and are displayed as relative activity (normalised to levels in cells transfected with 6PGD alone). Statistical analysis was performed using repeated measure one-way ANOVA followed by Dunnett's multiple comparison (ns, not significant; * P < 0.05; ** P < 0.01; **** P < 0.0001). ( B ) Protein expression of 6PGD (anti-Flag) and HDACs (anti-V5) in transfected HEK293 cells was assessed by immunoblotting, with GAPDH used as a loading control. The displayed blots are from one experiment, representative of two independent experiments. The red arrow indicates the shorter form of 6PGD (6PGD-s).

Article Snippet: Membranes were then probed with anti-Flag (mouse anti-Flag, 4 μg/ml, Sigma-Aldrich, F3165; rabbit anti-Flag, 0.8 μg/ml, Sigma-Aldrich, F7425; rabbit anti-Flag, 0.414 μg/ml, Cell Signaling Technology, 14793) or anti-V5 antibodies (mouse anti-V5, 1 μg/ml Bio-Rad, MCA1360; rabbit anti-V5, 0.192 μg/ml, Cell Signaling Technology, 13202) at 4°C overnight.

Techniques: Transfection, Expressing, Construct, Activity Assay, Comparison, Western Blot, Control