HDAC6 Antibody - #AF6485

Fig. 8: GRK5 bound to HDAC6 and promoted its phosphorylation. A The colocalization of GRK5 and HDAC6 in human chondrocytes was detected by immunofluorescence double staining (400×, scale bar: 50 µm). B Co-IP was used to determine the binding of GRK5 to HDAC6 in human chondrocytes. C The human GRK5 overexpressing plasmid with different domains (with a flag tag) was constructed and co-transfected with the human HDAC6 overexpressing plasmid (with a his tag) to 293T cells. After transfection for 48 h, the binding of GRK5 and HDAC6 was detected in Co-IP. D human chondrocytes were infected with GRK5 overexpressing plasmid (with flag tag) and kinase-inactive K215R GRK5 overexpressing plasmid (with flag tag), and western blotting was used to detect flag levels. E The levels of total HDAC6 and phosphorylated HDAC6 in chondrocytes were detected by western blotting. F The levels of COL2A1 and MMP13 were detected by real-time PCR. G Chondrocyte apoptosis was tested by TUNEL staining (200×, scale bar: 100 µm; arrows represented TUNEL-positive cells). H The levels of total HDAC6 and phosphorylated HDAC6 were determined in articular cartilage tissues (n = 6) and in chondrocytes (n = 3) by co-IP and western blotting. $, p 

Fig. 8: GRK5 bound to HDAC6 and promoted its phosphorylation. A The colocalization of GRK5 and HDAC6 in human chondrocytes was detected by immunofluorescence double staining (400×, scale bar: 50 µm). B Co-IP was used to determine the binding of GRK5 to HDAC6 in human chondrocytes. C The human GRK5 overexpressing plasmid with different domains (with a flag tag) was constructed and co-transfected with the human HDAC6 overexpressing plasmid (with a his tag) to 293T cells. After transfection for 48 h, the binding of GRK5 and HDAC6 was detected in Co-IP. D human chondrocytes were infected with GRK5 overexpressing plasmid (with flag tag) and kinase-inactive K215R GRK5 overexpressing plasmid (with flag tag), and western blotting was used to detect flag levels. E The levels of total HDAC6 and phosphorylated HDAC6 in chondrocytes were detected by western blotting. F The levels of COL2A1 and MMP13 were detected by real-time PCR. G Chondrocyte apoptosis was tested by TUNEL staining (200×, scale bar: 100 µm; arrows represented TUNEL-positive cells). H The levels of total HDAC6 and phosphorylated HDAC6 were determined in articular cartilage tissues (n = 6) and in chondrocytes (n = 3) by co-IP and western blotting. $, p 

Figure 1 HDAC6 knockdown enhances lysosomal acidification in SH-SY5Y cells.HDAC6 was knocked down in SH-SY5Y cells through transfection with shHDAC6. (A) Representative immunoblots for HDAC6, Ac-α-tubulin, and α-tubulin. (B, C) Relative protein expression levels based on band intensity (n = 3 independent experiments). (D) Immunofluorescence staining with antibodies against Ac-α-tubulin (green) and α-tubulin (red). Scale bar: 10 μm. (E) Quantitative analysis of relative Ac-α-tubulin expression levels (n = 30 cells in each group). (F) Representative immunoblots for V-ATPase subunits (ATP6V1A, ATP6V1C1, ATP6V0D) and Lamp2 in membrane fractions from cells subjected to HDAC6 knockdown. (G, H) Ratios of ATP6V1A and ATP6V1C1 to ATP6V0D, reflecting V-ATPase V1-V0 assembly (n = 3 independent experiments). (I) Relative ATP6V0D expression, normalized to Lamp2 expression, based on band density (n = 3 independent experiments). (J) Lamp2 immunofluorescence (red). Scale bar: 5 μm. (K, L) Size and number of Lamp2-positive puncta in cells transfected with shHDAC6 or shCTRL (n = 30 cells in each group). (M) LysoSensor yellow/blue DND-160 lysosomal acidification detection. Scale bar: 5 μm. (N) The degree of acidity is presented by the ratio of yellow to blue puncta (n = 30 cells in each group). (O) Calibration curve for lysosomal pH. (P) Lysosomal pH values in shCTRL and shHDAC6 SH-SY5Y cells (n = 5 independent cultures). *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student’s t-test). Ac-α-tubulin: Acetylated-α-tubulin; ex: excitation; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; HDAC6: histone deacetylase 6; ns: not significant; shHDAC6: HDAC6 shRNA.

FIGURE 6 HDAC6 inhibition restored HTRA1 expression in iAs-exposed Caco-2 cells. (A) RT-qPCR result showed the transcript level of HTRA1 in cells from the Ctrl and iAs groups treated with WT-161 (5 μM); DMSO was used as control. (B) The protein levels of HDAC6 and HTRA1 in response to WT-161 treatment were determined by Western blot analysis. (C) Apoptosis of cells treated with panobinostat (100 nM), WT-161 (5 μM), and mocetinostat (1 μM) for 48 h was examined by cytotoxicity assay. The sensitivity of the treatment of WT-161 (5 μM), 5-FU (6 μM) or the combination of these drugs for 48 h was analyzed by (D) cytotoxicity assay and (E) DNA fragmentation ELISA. Data were representatives of at least three independent experiments, shown as mean ± SD. The Significance threshold for one-way ANOVA:

Effect of emodin treatment on HDAC6 activity. (A) The modeled 3D structure of HDAC6 docked with emodin. (B) An enlarged view of the binding site is displayed in the inset box. (C) The interaction bonds of HDAC6 with emodin. The HDAC6 protein is shown in cyan, emodin is colored green, the interacting residues as red, bonds are shown as yellow dotted lines and bond lengths are depicted as numbers. (D) Representative immunofluorescence staining images and (E) quantitative intensity analysis of HDAC6 of each group. Scale bar, 20 µm. Representative (F) western blots and (G) quantitation of HDAC6 expression levels. Data are presented as the mean ± SD (n=5). *P