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BPS Bioscience hdac6 fluorogenic assay kit
Hdac6 Fluorogenic Assay Kit, supplied by BPS Bioscience, used in various techniques. Bioz Stars score: 94/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/hdac6+activity/HDAC6+Fluorogenic+Assay+Kit/custom%4050076%4040972425
Average 94 stars, based on 47 article reviews

hdac6 fluorogenic assay kit - by Bioz Stars, 2026-09

94/100 stars

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Clinical Proteomics:

Article Title: Cathepsin S Activity Controls Injury-Related Vascular Repair in Mice via the TLR2-Mediated p38MAPK and PI3K−Akt/p-HDAC6 Signaling Pathway
Article Snippet: .. 5 μl of protein samples and plasma was used to dectect HDAC6 activity by using the Fluorogenic HDAC6 Assay Kit (50076, BPS Bioscience, San Diego, CA) following the manufacturer’s instructions with a small modification. ..

Activity Assay:

Article Title: Substituted hydroxamic acids and uses thereof
Article Snippet: .. To measure the inhibition of HDAC6 activity, purified human HDAC6 (BPS Bioscience; Cat. No. 5006) is incubated with substrate Ac-Arg-Gly-Lys(Ac)-AMC peptide (Bachem Biosciences; Cat. No. 1-1925) for 1 hour at 30° C. in the presence of test compounds or vehicle DMSO control. .. The reaction is stopped with the HDAC inhibitor trichostatin A (Sigma; Cat. No. T8552) and the amount of Arg-Gly-Lys-AMC generated is quantitated by digestion with trypsin (Sigma; Cat. No. T1426) and subsequent measurement of the amount of AMC released using a fluorescent plate reader (Pherastar; BMG Technologies) set at Ex 340 nm and Em 460 nm.

Article Title: Substituted hydroxamic acids and uses thereof
Article Snippet: .. To measure the inhibition of HDAC6 activity, purified human HDAC6 (BPS Bioscience; Cat. No. 5006) is incubated with substrate Ac-Arg-Gly-Lys(Ac)-AMC peptide (Bachem Biosciences; Cat. No. I-1925) for 1 hour at 30° C. in the presence of test compounds or vehicle DMSO control. .. The reaction is stopped with the HDAC inhibitor trichostatin A (Sigma; Cat. No. T8552) and the amount of Arg-Gly-Lys-AMC generated is quantitated by digestion with trypsin (Sigma; Cat. No. T1426) and subsequent measurement of the amount of AMC released using a fluorescent plate reader (Pherastar, BMG Technologies) set at Ex 340 nm and Em 460 nm.

Article Title: Microtubule destabilization caused by particulate matter contributes to lung endothelial barrier dysfunction and inflammation
Article Snippet: .. HDAC6 activity was determined using a fluorogenic HDAC6 assay kit available from BPS Bioscience (San Diego, CA) following the manufacturer’s instructions. ..

Article Title: Staphylococcus aureus –induced endothelial permeability and inflammation are mediated by microtubule destabilization
Article Snippet: The cytokine levels in conditioned media from cells or BAL fluid from mouse lungs were determined using ELISA kits available from R&D Systems (Minneapolis, MN) according to the manufacturer's instructions. .. HDAC6 activity was measured using a fluorogenic HDAC6 assay kit available from BPS Bioscience (San Diego, CA) following the manufacturer's instructions. .. For knockdown of HDAC6, GEF-H1, or CLASP2 in human pulmonary EC cultures, predesigned human siRNA were ordered from Ambion (Austin, TX) in purified, desalted, deprotected, and annealed double-strand form.

Article Title: Substituted hydroxamic acids and uses thereof
Article Snippet: .. To measure the inhibition of HDAC6 activity, purified human HDAC6 (BPS Bioscience; Cat. No. 5006) is incubated with substrate Ac-Arg-Gly-Lys(Ac)-AMC peptide (Bachem Biosciences; Cat. No. I-1925) for 1 hour at 30° C. in the presence of test compounds or vehicle DMSO control. .. The reaction is stopped with the HDAC inhibitor trichostatin A (Sigma; Cat. No. T8552) and the amount of Arg-Gly-Lys-AMC generated is quantitated by digestion with trypsin (Sigma; Cat. No. T1426) and subsequent measurement of the amount of AMC released using a fluorescent plate reader (Pherastar; BMG Technologies) set at Ex 340 nm and Em 460 nm.

Article Title: Autophagic LC3 + calcified extracellular vesicles initiate cartilage calcification in osteoarthritis
Article Snippet: After treatment with the primary antibodies, the blotted membrane was incubated with secondary antibodies and visualized using a chemiluminescence detection system (Chemidoc, Bio-Rad, XRS, Hercules, CA, USA). .. Evaluation of HDAC6 activity was conducted according to the manufacturer’s instructions (#50076, BPS Bioscience, San Diego, CA, USA). ..

Modification:

Inhibition:

Article Title: Substituted hydroxamic acids and uses thereof
Article Snippet: .. To measure the inhibition of HDAC6 activity, purified human HDAC6 (BPS Bioscience; Cat. No. 5006) is incubated with substrate Ac-Arg-Gly-Lys(Ac)-AMC peptide (Bachem Biosciences; Cat. No. I-1925) for 1 hour at 30° C. in the presence of test compounds or vehicle DMSO control. .. The reaction is stopped with the HDAC inhibitor trichostatin A (Sigma; Cat. No. T8552) and the amount of Arg-Gly-Lys-AMC generated is quantitated by digestion with trypsin (Sigma; Cat. No. T1426) and subsequent measurement of the amount of AMC released using a fluorescent plate reader (Pherastar, BMG Technologies) set at Ex 340 nm and Em 460 nm.

Article Title: Substituted hydroxamic acids and uses thereof
Article Snippet: .. To measure the inhibition of HDAC6 activity, purified human HDAC6 (BPS Bioscience; Cat. No. 5006) is incubated with substrate Ac-Arg-Gly-Lys(Ac)-AMC peptide (Bachem Biosciences; Cat. No. I-1925) for 1 hour at 30° C. in the presence of test compounds or vehicle DMSO control. .. The reaction is stopped with the HDAC inhibitor trichostatin A (Sigma; Cat. No. T8552) and the amount of Arg-Gly-Lys-AMC generated is quantitated by digestion with trypsin (Sigma; Cat. No. T1426) and subsequent measurement of the amount of AMC released using a fluorescent plate reader (Pherastar; BMG Technologies) set at Ex 340 nm and Em 460 nm.

Purification:

Article Title: Substituted hydroxamic acids and uses thereof
Article Snippet: .. To measure the inhibition of HDAC6 activity, purified human HDAC6 (BPS Bioscience; Cat. No. 5006) is incubated with substrate Ac-Arg-Gly-Lys(Ac)-AMC peptide (Bachem Biosciences; Cat. No. I-1925) for 1 hour at 30° C. in the presence of test compounds or vehicle DMSO control. .. The reaction is stopped with the HDAC inhibitor trichostatin A (Sigma; Cat. No. T8552) and the amount of Arg-Gly-Lys-AMC generated is quantitated by digestion with trypsin (Sigma; Cat. No. T1426) and subsequent measurement of the amount of AMC released using a fluorescent plate reader (Pherastar, BMG Technologies) set at Ex 340 nm and Em 460 nm.

Article Title: Substituted hydroxamic acids and uses thereof
Article Snippet: .. To measure the inhibition of HDAC6 activity, purified human HDAC6 (BPS Bioscience; Cat. No. 5006) is incubated with substrate Ac-Arg-Gly-Lys(Ac)-AMC peptide (Bachem Biosciences; Cat. No. I-1925) for 1 hour at 30° C. in the presence of test compounds or vehicle DMSO control. .. The reaction is stopped with the HDAC inhibitor trichostatin A (Sigma; Cat. No. T8552) and the amount of Arg-Gly-Lys-AMC generated is quantitated by digestion with trypsin (Sigma; Cat. No. T1426) and subsequent measurement of the amount of AMC released using a fluorescent plate reader (Pherastar; BMG Technologies) set at Ex 340 nm and Em 460 nm.

Incubation:

Article Title: Substituted hydroxamic acids and uses thereof
Article Snippet: .. To measure the inhibition of HDAC6 activity, purified human HDAC6 (BPS Bioscience; Cat. No. 5006) is incubated with substrate Ac-Arg-Gly-Lys(Ac)-AMC peptide (Bachem Biosciences; Cat. No. I-1925) for 1 hour at 30° C. in the presence of test compounds or vehicle DMSO control. .. The reaction is stopped with the HDAC inhibitor trichostatin A (Sigma; Cat. No. T8552) and the amount of Arg-Gly-Lys-AMC generated is quantitated by digestion with trypsin (Sigma; Cat. No. T1426) and subsequent measurement of the amount of AMC released using a fluorescent plate reader (Pherastar, BMG Technologies) set at Ex 340 nm and Em 460 nm.

Article Title: Substituted hydroxamic acids and uses thereof
Article Snippet: .. To measure the inhibition of HDAC6 activity, purified human HDAC6 (BPS Bioscience; Cat. No. 5006) is incubated with substrate Ac-Arg-Gly-Lys(Ac)-AMC peptide (Bachem Biosciences; Cat. No. I-1925) for 1 hour at 30° C. in the presence of test compounds or vehicle DMSO control. .. The reaction is stopped with the HDAC inhibitor trichostatin A (Sigma; Cat. No. T8552) and the amount of Arg-Gly-Lys-AMC generated is quantitated by digestion with trypsin (Sigma; Cat. No. T1426) and subsequent measurement of the amount of AMC released using a fluorescent plate reader (Pherastar; BMG Technologies) set at Ex 340 nm and Em 460 nm.

Control:

Article Title: Substituted hydroxamic acids and uses thereof
Article Snippet: .. To measure the inhibition of HDAC6 activity, purified human HDAC6 (BPS Bioscience; Cat. No. 5006) is incubated with substrate Ac-Arg-Gly-Lys(Ac)-AMC peptide (Bachem Biosciences; Cat. No. I-1925) for 1 hour at 30° C. in the presence of test compounds or vehicle DMSO control. .. The reaction is stopped with the HDAC inhibitor trichostatin A (Sigma; Cat. No. T8552) and the amount of Arg-Gly-Lys-AMC generated is quantitated by digestion with trypsin (Sigma; Cat. No. T1426) and subsequent measurement of the amount of AMC released using a fluorescent plate reader (Pherastar, BMG Technologies) set at Ex 340 nm and Em 460 nm.

Article Title: Substituted hydroxamic acids and uses thereof
Article Snippet: .. To measure the inhibition of HDAC6 activity, purified human HDAC6 (BPS Bioscience; Cat. No. 5006) is incubated with substrate Ac-Arg-Gly-Lys(Ac)-AMC peptide (Bachem Biosciences; Cat. No. I-1925) for 1 hour at 30° C. in the presence of test compounds or vehicle DMSO control. .. The reaction is stopped with the HDAC inhibitor trichostatin A (Sigma; Cat. No. T8552) and the amount of Arg-Gly-Lys-AMC generated is quantitated by digestion with trypsin (Sigma; Cat. No. T1426) and subsequent measurement of the amount of AMC released using a fluorescent plate reader (Pherastar; BMG Technologies) set at Ex 340 nm and Em 460 nm.

Image Search Results

Representative HDAC6 PET tracers with IC 50 values for HDAC6.

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of Pyrazole-Based Positron Emission Tomography Agent that Maps Histone Deacetylase 6 (HDAC6) in the Nonhuman Primate Brain

doi: 10.1021/acs.jmedchem.5c02216

Figure Lengend Snippet: Representative HDAC6 PET tracers with IC 50 values for HDAC6.

Article Snippet: The binding assay using AS-MS was performed at rt in a final volume of 10 μL using the recombinant HDAC6 (HDAC6, Active, H88-30G-10, Lot:B2010-9; SignalChem Biotech Inc., Richmond, BC, Canada).

Techniques:

Affinity selection (AS)–MS binding study of HDAC6 with 16a and bavarostat. Data points are the mean ± standard error (SE) (four technical replicates). (A) Saturation binding curve. (B) Time course for the dissociation. (C) Binding parameters calculated from AS–MS data. Functional IC 50 values are shown for comparison.

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of Pyrazole-Based Positron Emission Tomography Agent that Maps Histone Deacetylase 6 (HDAC6) in the Nonhuman Primate Brain

doi: 10.1021/acs.jmedchem.5c02216

Figure Lengend Snippet: Affinity selection (AS)–MS binding study of HDAC6 with 16a and bavarostat. Data points are the mean ± standard error (SE) (four technical replicates). (A) Saturation binding curve. (B) Time course for the dissociation. (C) Binding parameters calculated from AS–MS data. Functional IC 50 values are shown for comparison.

Techniques: Selection, Binding Assay, Functional Assay, Comparison

Brain and plasma concentration of 16a in wild-type (WT) and HDAC6 knockout (KO) mice. Compound 16a (1 mg/kg) was intravenously administered to WT and HDAC6 KO mice. Two hours after administration, the hippocampus and plasma were collected and the concentration of 16a in the brain (A) and plasma (B) was measured using liquid chromatography with tandem mass spectrometry (LC–MS/MS). The data are expressed in percentage of injected dose per gram of brain tissue (%ID/g) or per milliliter of plasma (%ID/mL), respectively, as mean ± standard error (SE), n = 4. ** P < 0.01, n.s.: not significant.

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of Pyrazole-Based Positron Emission Tomography Agent that Maps Histone Deacetylase 6 (HDAC6) in the Nonhuman Primate Brain

doi: 10.1021/acs.jmedchem.5c02216

Figure Lengend Snippet: Brain and plasma concentration of 16a in wild-type (WT) and HDAC6 knockout (KO) mice. Compound 16a (1 mg/kg) was intravenously administered to WT and HDAC6 KO mice. Two hours after administration, the hippocampus and plasma were collected and the concentration of 16a in the brain (A) and plasma (B) was measured using liquid chromatography with tandem mass spectrometry (LC–MS/MS). The data are expressed in percentage of injected dose per gram of brain tissue (%ID/g) or per milliliter of plasma (%ID/mL), respectively, as mean ± standard error (SE), n = 4. ** P < 0.01, n.s.: not significant.

Techniques: Clinical Proteomics, Concentration Assay, Knock-Out, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Injection

Comparison of HDAC6 expression levels in a mouse and monkey using Western blotting. Hippocampus (Hip) was obtained from two mice, and HDAC6 expression was examined. Recombinant HDAC6 conjugated with GST (0.03, 0.1, 0.3, and 1 ng) was also used for the positive controls (A). Lysates of the hippocampus and corpus callosum (CC) obtained from a monkey were applied to Western blotting in duplicate. The same controls were used for comparison (B).

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of Pyrazole-Based Positron Emission Tomography Agent that Maps Histone Deacetylase 6 (HDAC6) in the Nonhuman Primate Brain

doi: 10.1021/acs.jmedchem.5c02216

Figure Lengend Snippet: Comparison of HDAC6 expression levels in a mouse and monkey using Western blotting. Hippocampus (Hip) was obtained from two mice, and HDAC6 expression was examined. Recombinant HDAC6 conjugated with GST (0.03, 0.1, 0.3, and 1 ng) was also used for the positive controls (A). Lysates of the hippocampus and corpus callosum (CC) obtained from a monkey were applied to Western blotting in duplicate. The same controls were used for comparison (B).

Techniques: Comparison, Expressing, Western Blot, Recombinant

Immunohistochemical (IHC) staining of HDAC6 protein in a monkey brain. Coronal brain sections, including the areas below were prepared from a monkey (a, putamen; b, caudate nucleus; c, corpus callosum; d, white matter; e, anterior cingulate cortex; f, thalamus; g, hippocampus; h, cerebellum; i, brain stem [pons]). They were immunostained with HDAC6 antibody.

Journal: Journal of Medicinal Chemistry

Article Title: Discovery of Pyrazole-Based Positron Emission Tomography Agent that Maps Histone Deacetylase 6 (HDAC6) in the Nonhuman Primate Brain

doi: 10.1021/acs.jmedchem.5c02216

Figure Lengend Snippet: Immunohistochemical (IHC) staining of HDAC6 protein in a monkey brain. Coronal brain sections, including the areas below were prepared from a monkey (a, putamen; b, caudate nucleus; c, corpus callosum; d, white matter; e, anterior cingulate cortex; f, thalamus; g, hippocampus; h, cerebellum; i, brain stem [pons]). They were immunostained with HDAC6 antibody.

Techniques: Immunohistochemical staining, Immunohistochemistry

( A ) Ligand-based pharmacophore model derived from a diverse set of known HDAC6 inhibitors, highlighting key pharmacophoric features: hydrogen bond donors (green spheres), hydrogen bond acceptors (red spheres), hydrophobic regions (yellow sphere), and ionizable groups (red arrows). ( B ) Receiver Operating Characteristic (ROC) curve generated by automated analysis in LigandScout , demonstrating the model’s ability to discriminate between active and inactive compounds.

Journal: Pharmaceuticals

Article Title: Discovery of a Promising Hydroxyamino-Piperidine HDAC6 Inhibitor via Integrated Virtual Screening and Experimental Validation in Multiple Myeloma

doi: 10.3390/ph18091303

Figure Lengend Snippet: ( A ) Ligand-based pharmacophore model derived from a diverse set of known HDAC6 inhibitors, highlighting key pharmacophoric features: hydrogen bond donors (green spheres), hydrogen bond acceptors (red spheres), hydrophobic regions (yellow sphere), and ionizable groups (red arrows). ( B ) Receiver Operating Characteristic (ROC) curve generated by automated analysis in LigandScout , demonstrating the model’s ability to discriminate between active and inactive compounds.

Article Snippet: HDAC6 enzymatic activity was assessed using a fluorogenic HDAC6 assay kit (BPS Bioscience, cat#50076, San Diego, CA, USA), which includes a fluorometric substrate containing an acetylated lysine side chain.

Techniques: Derivative Assay, Generated

Workflow of the virtual screening process for discovering novel HDAC6 inhibitors, accompanied by the 2D structures of four selected compounds prioritized for experimental validation.

Journal: Pharmaceuticals

Article Title: Discovery of a Promising Hydroxyamino-Piperidine HDAC6 Inhibitor via Integrated Virtual Screening and Experimental Validation in Multiple Myeloma

doi: 10.3390/ph18091303

Figure Lengend Snippet: Workflow of the virtual screening process for discovering novel HDAC6 inhibitors, accompanied by the 2D structures of four selected compounds prioritized for experimental validation.

Techniques: Biomarker Discovery

Three-dimensional representations of the best docking poses for the four hit compounds selected from the virtual screening. The binding conformations within the HDAC6 active site are shown for: Compound 7 (AKOS030496586), Compound 8 (AKOS000531201), Compound 9 (AKOS030461429) and Compound 10 (AKOS030273637). Ligands are represented as yellow carbon sticks, the protein is depicted as a mauve ribbon, and the catalytic zinc ion is shown as a gray sphere. Key interacting residues are highlighted to illustrate binding interactions.

Journal: Pharmaceuticals

Article Title: Discovery of a Promising Hydroxyamino-Piperidine HDAC6 Inhibitor via Integrated Virtual Screening and Experimental Validation in Multiple Myeloma

doi: 10.3390/ph18091303

Figure Lengend Snippet: Three-dimensional representations of the best docking poses for the four hit compounds selected from the virtual screening. The binding conformations within the HDAC6 active site are shown for: Compound 7 (AKOS030496586), Compound 8 (AKOS000531201), Compound 9 (AKOS030461429) and Compound 10 (AKOS030273637). Ligands are represented as yellow carbon sticks, the protein is depicted as a mauve ribbon, and the catalytic zinc ion is shown as a gray sphere. Key interacting residues are highlighted to illustrate binding interactions.

Techniques: Binding Assay

Journal: Pharmaceuticals

Article Title: Discovery of a Promising Hydroxyamino-Piperidine HDAC6 Inhibitor via Integrated Virtual Screening and Experimental Validation in Multiple Myeloma

doi: 10.3390/ph18091303

Figure Lengend Snippet: MDs and MM-GBSA analysis of Compound 8 , Compound 10 , Compound 9 , Compound 7 , and Trichostatin A (used as control) in complex with HDAC6. ( A ) RMSD of ligand heavy atoms over the simulation, expressed in Å; ( B ) Protein-ligand interaction profile showing contacts between ligand atoms and HDAC6 residues. Only interactions occurring for more than 30% of the 200 ns simulation time are displayed; ( C ) Binding free energy profiles calculated using the MM-GBSA method throughout the simulation.

Techniques: Control, Binding Assay

A. Representative immunofluorescent images of 3- and 6-month MCOs derived from SPAST WT/WT , SPAST WT/S245X and SPAST WT/C448Y lines, untreated or treated with the HDAC6 inhibitor Tub A, stained for acetylated tubulin and βIII-tubulin. B-C. Quantification of the acetylated tubulin to βIII-tubulin ratio in 3-month (B) and 6-month (C) MCOs (n=3-6). Tub A significantly increased tubulin acetylation in mutant MCOs. D. Representative immunofluorescent images of 3- and 6-month-old MCOs from SPAST WT/WT , SPAST WT/S245X and SPAST WT/C448Y lines, untreated or treated with TubA, stained for SMI32 (a marker of early axonal degeneration) and βIII-tubulin. E-F. Quantification of the SMI32 to βIII-tubulin ratio in individual axons from 3-month (n=8, E) and 6-month (n=10, F) MCOs. Tub A treatment significantly reduced SMI32 levels in mutant organoids. G-H. Quantification of HDAC6 enzymatic activity in 3-month (n=3, G) and 6-month (n=3, H) MCOs from SPAST WT/WT , SPAST WT/S245X and SPAST WT/C448Y lines, with or without Tub A treatment. HDAC6 activity is expressed as units (U) per mg of total protein, where one unit is defined as the amount of enzyme required to deacetylate 1pmol of a synthetic acetylated peptide substrate per minute. Elevated HDAC6 activity in mutant organoids is significantly reduced by Tub A treatment at both time points. I-J. WB (I) and quantification (J) of HDAC6 (black arrow) protein levels in 3-month MCOs derived from SPAST WT/WT , SPAST WT/S245X and SPAST WT/C448Y lines. HDAC6 expression, normalized to GAPDH and shown relative to SPAST WT/WT , revealed no significant differences across genotypes (n = 4). One-way ANOVA with Tukey post hoc analysis. *p<0.05, **p<0.002, ***p<0.001, ****p<0.0001. All data shown as mean ± SD, see Supplementary Tables 3-4 for details.

Journal: bioRxiv

Article Title: Genotype–Phenotype Distinctions in Spastic Paraplegia 4 Reveal HDAC6 as a Therapeutic Target

doi: 10.1101/2025.07.15.664947

Figure Lengend Snippet: A. Representative immunofluorescent images of 3- and 6-month MCOs derived from SPAST WT/WT , SPAST WT/S245X and SPAST WT/C448Y lines, untreated or treated with the HDAC6 inhibitor Tub A, stained for acetylated tubulin and βIII-tubulin. B-C. Quantification of the acetylated tubulin to βIII-tubulin ratio in 3-month (B) and 6-month (C) MCOs (n=3-6). Tub A significantly increased tubulin acetylation in mutant MCOs. D. Representative immunofluorescent images of 3- and 6-month-old MCOs from SPAST WT/WT , SPAST WT/S245X and SPAST WT/C448Y lines, untreated or treated with TubA, stained for SMI32 (a marker of early axonal degeneration) and βIII-tubulin. E-F. Quantification of the SMI32 to βIII-tubulin ratio in individual axons from 3-month (n=8, E) and 6-month (n=10, F) MCOs. Tub A treatment significantly reduced SMI32 levels in mutant organoids. G-H. Quantification of HDAC6 enzymatic activity in 3-month (n=3, G) and 6-month (n=3, H) MCOs from SPAST WT/WT , SPAST WT/S245X and SPAST WT/C448Y lines, with or without Tub A treatment. HDAC6 activity is expressed as units (U) per mg of total protein, where one unit is defined as the amount of enzyme required to deacetylate 1pmol of a synthetic acetylated peptide substrate per minute. Elevated HDAC6 activity in mutant organoids is significantly reduced by Tub A treatment at both time points. I-J. WB (I) and quantification (J) of HDAC6 (black arrow) protein levels in 3-month MCOs derived from SPAST WT/WT , SPAST WT/S245X and SPAST WT/C448Y lines. HDAC6 expression, normalized to GAPDH and shown relative to SPAST WT/WT , revealed no significant differences across genotypes (n = 4). One-way ANOVA with Tukey post hoc analysis. *p<0.05, **p<0.002, ***p<0.001, ****p<0.0001. All data shown as mean ± SD, see Supplementary Tables 3-4 for details.

Article Snippet: HDAC6 activity assays were performed according to the manufacturer’s instructions (BPS Biosciences, 50076-1) and as previously described., Briefly, treated and untreated organoid samples, as well as spinal cord tissue samples, were lysed in HDCA6 lysis buffer and centrifuged at 16,000g for 10 mins at 4°C.

Techniques: Derivative Assay, Staining, Mutagenesis, Marker, Activity Assay, Expressing

A-B. Co-immunoprecipitation (Co-IP) in HEK293T cells co-expressing HDAC6-mCherry and Flag-tagged spastin isoforms revealed that HDAC6 binds selectively to M1-spastin, but not M87-spastin (A). Quantification confirmed robust enrichment of M1-spastin isoforms, in the HDAC6-containing immunoprecipitated complex, with significantly increased interaction observed for mutant forms (B). C-D. Reciprocal Co-IP using anti-Flag antibody was performed to isolate spastin isoforms, followed by WB with anti-HDAC6 and anti-phospho-HDAC6 antibodies, showing M1-Spastin C448Y -Flag immunoprecipitated more phosphorylated HDAC6. (C). Quantification also confirmed that mutant M1-spastin isoforms pulled down greater amounts of HDAC6 compared to controls (D). E. HDAC6 activity assays in SH-SY5Y cells expressing various M1 and M87 spastin mutants showed that only M1 mutations led to significantly elevated HDAC6 enzymatic activity, indicating an isoform-specific gain-of-function effect. One-way ANOVA with Tukey post hoc analysis. **p<0.002, ***p<0.001, and Unpaired t-test. ##p<0.005, ###p<0.001. All data shown as mean ± SD, see Supplementary Tables 5-6 for details.

Journal: bioRxiv

Article Title: Genotype–Phenotype Distinctions in Spastic Paraplegia 4 Reveal HDAC6 as a Therapeutic Target

doi: 10.1101/2025.07.15.664947

Figure Lengend Snippet: A-B. Co-immunoprecipitation (Co-IP) in HEK293T cells co-expressing HDAC6-mCherry and Flag-tagged spastin isoforms revealed that HDAC6 binds selectively to M1-spastin, but not M87-spastin (A). Quantification confirmed robust enrichment of M1-spastin isoforms, in the HDAC6-containing immunoprecipitated complex, with significantly increased interaction observed for mutant forms (B). C-D. Reciprocal Co-IP using anti-Flag antibody was performed to isolate spastin isoforms, followed by WB with anti-HDAC6 and anti-phospho-HDAC6 antibodies, showing M1-Spastin C448Y -Flag immunoprecipitated more phosphorylated HDAC6. (C). Quantification also confirmed that mutant M1-spastin isoforms pulled down greater amounts of HDAC6 compared to controls (D). E. HDAC6 activity assays in SH-SY5Y cells expressing various M1 and M87 spastin mutants showed that only M1 mutations led to significantly elevated HDAC6 enzymatic activity, indicating an isoform-specific gain-of-function effect. One-way ANOVA with Tukey post hoc analysis. **p<0.002, ***p<0.001, and Unpaired t-test. ##p<0.005, ###p<0.001. All data shown as mean ± SD, see Supplementary Tables 5-6 for details.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Mutagenesis, Activity Assay

A. Experimental timeline of daily Tub A or vehicle treatment and CatWalk analysis in WT and dHet mice. B. Representative CatWalk print view showing paw positioning and parameters analyzed, including print length, width, area, and position. C–F. Quantitative gait metrics across experimental groups: Print length (C), Print width (D), Print area (E), and Print position (F) (hind paw placement relative to the ipsilateral front paw). Data represent classified runs from 6 animals per group. G. HDAC6 enzymatic activity measured from spinal cord lysates across groups (n=3 mice per group). H-J. WB analysis of lumbar spinal cord lysates showing levels of acetylated tubulin and βIII-tubulin in WT and dHet mice treated with Tub A or vehicle. Tub A treatment significantly increases acetylated tubulin levels in dHet mice. βIII-tubulin levels remain unchanged across groups and serve as a loading control (n=3 per group). K. Representative toluidine blue-stained cross-sections of the dorsal column in lumbar spinal cords from treated and untreated WT and dHet mice. L. Quantitative analyses of CST axons: total axon count per μm² (L), average axonal perimeter (M), and number of morphologically normal versus swollen axons (based on shape classification as normal: regular, swollen: irregular (N). Axons with a perimeter of ≥ 10µm was considered swollen). Data represents cumulative axon counts (n=120-500) from 3 mice per group. One-way ANOVA with Tukey post hoc analysis. *p<0.05, **p<0.002, ***p<0.001, ****p<0.0001 and Unpaired t-test. #p<0.05, ##p<0.005. All data shown as mean ± SD, see Supplementary Table 8 for details.

Journal: bioRxiv

Article Title: Genotype–Phenotype Distinctions in Spastic Paraplegia 4 Reveal HDAC6 as a Therapeutic Target

doi: 10.1101/2025.07.15.664947

Figure Lengend Snippet: A. Experimental timeline of daily Tub A or vehicle treatment and CatWalk analysis in WT and dHet mice. B. Representative CatWalk print view showing paw positioning and parameters analyzed, including print length, width, area, and position. C–F. Quantitative gait metrics across experimental groups: Print length (C), Print width (D), Print area (E), and Print position (F) (hind paw placement relative to the ipsilateral front paw). Data represent classified runs from 6 animals per group. G. HDAC6 enzymatic activity measured from spinal cord lysates across groups (n=3 mice per group). H-J. WB analysis of lumbar spinal cord lysates showing levels of acetylated tubulin and βIII-tubulin in WT and dHet mice treated with Tub A or vehicle. Tub A treatment significantly increases acetylated tubulin levels in dHet mice. βIII-tubulin levels remain unchanged across groups and serve as a loading control (n=3 per group). K. Representative toluidine blue-stained cross-sections of the dorsal column in lumbar spinal cords from treated and untreated WT and dHet mice. L. Quantitative analyses of CST axons: total axon count per μm² (L), average axonal perimeter (M), and number of morphologically normal versus swollen axons (based on shape classification as normal: regular, swollen: irregular (N). Axons with a perimeter of ≥ 10µm was considered swollen). Data represents cumulative axon counts (n=120-500) from 3 mice per group. One-way ANOVA with Tukey post hoc analysis. *p<0.05, **p<0.002, ***p<0.001, ****p<0.0001 and Unpaired t-test. #p<0.05, ##p<0.005. All data shown as mean ± SD, see Supplementary Table 8 for details.

Techniques: Activity Assay, Control, Staining

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hdac6 fluorogenic assay kit (BPS Bioscience)

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