hdac5 s259 498a mutant cdna  (Addgene inc)

Journal: Nature communications

Article Title: A FAK/HDAC5 signaling axis controls osteocyte mechanotransduction.

doi: 10.1038/s41467-020-17099-3

Figure Lengend Snippet: Fig. 5 FAK-mediated class IIa HDACs tyrosine phosphorylation is regulated by FFSS. a Immunoprecipitation by anti-phosphotyrosine (referred to as “p- Y-1000” throughout) antibody. FFSS was performed for 10 min with Ocy454 cells. Phosphotyrosine immunoprecipitation was performed on cell lysates treated as indicated, and protein expression was determined by western blotting. FFSS reduced HDAC4 and HDAC5 with p-Y-1000 immunoprecipitation. b FLAG immunoprecipitation was performed in HDAC5-deficient cells stably expressing FLAG-HDAC5, subjected to FFSS for times as indicated. phospho- HDAC5 was decreased in a time-dependent manner. c HEK293T cells were transfected with FLAG-HDAC4, then treated for one hour as indicated with PF562271 (10 µM) or vanadate (1 mM) followed by phosphotyrosine immunoprecipitation and then immunoblotting. Tyrosine-phosphorylated HDAC4 was increased by vanadate treatment and decreased by FAK inhibitor treatment. P-values adjusted for multiple comparisons vs control are shown. d FAK kinase activity was measured by ADP-Glo kinase assay. E4Y1 (poly(Glu)/poly(Tyr) ratio of 4:1) polypeptides were used as a control substrate of FAK tyrosine kinase. Increased FAK kinase activity was detected when recombinant human HDAC5 protein was used as a substrate. e Recombinant human HDAC5 and E4Y1 (a control substrate) were incubated with ɣ-32P-ATP and FAK tyrosine kinase for 1 h, then separate by SDS-PAGE followed by autoradiography. FAK treatment showed ɣ-32P-ATP-positive band at the expected HDAC5 recombinant protein size. f Kinase assay reactions as in e were separated by SDS- PAGE followed by immunoblotting as indicated. CBB indicates Coomassie Brilliant Blue stain. One-way ANOVA followed by Tukey–Kramer post hoc test was used (d). Data are expressed as mean ± SEM. Each experiment was repeated three times (a–c, e, f). Source data are provided as a Source Data file.

Article Snippet: HDAC5 S259/498A mutant cDNA was obtained from Addgene (plasmid 32216), HDAC5 Y642F construct was synthesized de novo (VectorBuilder).

Techniques: Phospho-proteomics, Immunoprecipitation, Expressing, Western Blot, Stable Transfection, Transfection, Control, Activity Assay, Kinase Assay, Recombinant, Incubation, SDS Page, Autoradiography, Staining

Journal: Nature communications

Article Title: A FAK/HDAC5 signaling axis controls osteocyte mechanotransduction.

doi: 10.1038/s41467-020-17099-3

Figure Lengend Snippet: Fig. 7 RGD peptide blocks FAK activity and reduce Sost in a HDAC4/5-dependent manner. a Ocy454 cells were treated with cilengitide (10 µM and 50 µM) or PF562271 (10 µM) for 4 h, followed by RT-qPCR for FFSS-regulated genes as indicated. Both cilengitide and PF562271 regulate expression of FFSS-responsive genes. (n = 4 biologic replicates for RNA) P-values adjusted for multiple comparisons vs controls are shown. b Cells were treated with cilengitide (50 µM) for the indicated times followed by immunoblotting. c Cells were treated with the indicated doses of cilengitide (1 h) followed by immunoblotting. pHDAC4/5 immunoblotting was performed using an antibody that recognizes HDAC4 pS246 and HDAC5 pS259. d Saos2 cells were treated with cilengitide (100 µM) for 60 min followed by immunoblotting. e WT and HDAC4/5 double-knockout (H4H5-DKO) cells were treated with vehicle or cilengitide (50 µM) for 4 h followed by Sost RT-qPCR. Cilengitide treatment decreased Sost expression in control cells, but not in H4H5-DKO cells. (n = 4 biologic replicates for RNA) P-values vs control in the left and right panels. Two-sided unpaired t test e and one-way ANOVA followed by Tukey–Kramer post hoc test (a) were used. Data are expressed as mean ± SEM. Each experiment was repeated three times (b–d). Source data are provided as a Source Data file.

Article Snippet: HDAC5 S259/498A mutant cDNA was obtained from Addgene (plasmid 32216), HDAC5 Y642F construct was synthesized de novo (VectorBuilder).

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Western Blot, Double Knockout, Control