hdac6 d2e5 rabbit mab  (Cell Signaling Technology Inc)

Journal: Frontiers in Physiology

Article Title: Endothelial cell elongation and alignment in response to shear stress requires acetylation of microtubules

doi: 10.3389/fphys.2024.1425620

Figure Lengend Snippet: Shear stress alters post-translational modifications under shear stress Protein expression of PTMs at static (48 h), 0.5, 8, 24 and 48 h of shear stress. (A) Detyrosination (B) Polyglutamylation (C) Tyrosination (D) Acetylation. One sample Wilcoxon test. n = at least 3 technical replications with 3 biological replicates. (E) Transcriptomic data showing mRNA levels of various genes over 48 h of shear stress. Mann-Whitney U test. n = 4 biological replicates. (F) Protein expression of HDAC6 levels at indicated time course of static and laminar flow. Proteins were normalized to GAPDH. n = 5 technical replicates. One samplet-test. Data shown as mean ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: Primary antibodies monoclonal rabbit Acetylated -α-tubulin (Lys40) (D20G3) (Cell Signaling #5335, RRID:AB_10544694, 1:1,000), monoclonal mouse anti-Acetylated α-tubulin (Millipore Sigma #T7451, RRID:AB_609894, 1:1,000), monoclonal mouse anti-Acetylated (Lys40) (6-11B-1) α-tubulin (Cell Signaling #12152s, RRID:AB_2797830, 1:1,000), monoclonal mouse DM1a (Cell Signaling #3873s, RRID:AB_1904178, 1:1,000), polyclonal rabbit anti-detyrosinated tubulin (Millipore Sigma #AB3201, RRID:AB_117350 1:1,000), monoclonal mouse anti-polyglutamylated tubulin, clone B3 (Sigma-Aldrich #T9822,RRID:AB_477598, 1:1,000), monoclonal rat Anti-Tubulin Antibody, clone YL1/2 (Tyrosinated-tubulin) (Millipore Sigma #MAB1864, RRID:AB_1679330, 1:1,000), mouse monoclonal GAPDH (Millipore Sigma #MAB374, RRID:AB_2107445, 1:1,000), monoclonal rat α-tubulin (YOL1/34) (Invitrogen #MA1-80189, RRID:AB_2210200, 1:1,000) and monoclonal rabbit HDAC6 (D2E5) antibody (Cell Signaling #7558s, RRID:AB_10891804, 1:1,000) were used.

Techniques: Shear, Expressing, MANN-WHITNEY

Journal: Frontiers in Physiology

Article Title: Endothelial cell elongation and alignment in response to shear stress requires acetylation of microtubules

doi: 10.3389/fphys.2024.1425620

Figure Lengend Snippet: Genetic inactivation of HDAC6 and αTAT1 leads to altered acetylation levels and disruptions of cell shape under shear stress. (A) Immunofluorescence images of acetylation and total tubulin in control, HDAC6KO, and αTAT1KO human umbilical vein endothelial cells (HUVECs) under static and flow. Scale bar: 20 um. Yellow arrows point to the increased acetylation seen in control ECs under shear stress. (B) Radial graphs comparing control vs KO HUVECs under static and shear stress. n = 100 per condition, 2 biological replicates. Coefficients of variations for static (first column) are 0.57, 0.52, 0.56 and for flow (second column) are 0.23, 0.53 and 0.5 . (C) Elongation factor comparing control and KO HUVECs. n = 300 per condition, 2 biological replicates. Mann-Whitney U test. (D) Protein expression of α-tubulin, acetylated α-tubulin and GAPDH comparing control and αTAT1KO HUVECs. (E) Quantification of acetylation between control and aTAT1KO HUVEC. n = 16 technical replicates, 3 biological replicates. One sample Wilcoxon test. (F) Immunoblots of HDAC6, α-tubulin, acetylated α-tubulin and GAPDH comparing control and HDAC6KO HUVECs. (G) Protein expression of HDAC6 between control and HDAC6KO; n = 18 technical replicates, 3 biological replicates. One sample Wilcoxon test. (H) Protein expression of acetylated α-tubulin from control compared to HDAC6KO HUVECs. n = 14 technical replicates, 3 biological replicates. One sample Wilcoxon test. Data shown as mean ± SD, except (D) which is shown at mean ± SEM. * p <0.05 ** p < 0.01, *** p < 0.0001.

Article Snippet: Primary antibodies monoclonal rabbit Acetylated -α-tubulin (Lys40) (D20G3) (Cell Signaling #5335, RRID:AB_10544694, 1:1,000), monoclonal mouse anti-Acetylated α-tubulin (Millipore Sigma #T7451, RRID:AB_609894, 1:1,000), monoclonal mouse anti-Acetylated (Lys40) (6-11B-1) α-tubulin (Cell Signaling #12152s, RRID:AB_2797830, 1:1,000), monoclonal mouse DM1a (Cell Signaling #3873s, RRID:AB_1904178, 1:1,000), polyclonal rabbit anti-detyrosinated tubulin (Millipore Sigma #AB3201, RRID:AB_117350 1:1,000), monoclonal mouse anti-polyglutamylated tubulin, clone B3 (Sigma-Aldrich #T9822,RRID:AB_477598, 1:1,000), monoclonal rat Anti-Tubulin Antibody, clone YL1/2 (Tyrosinated-tubulin) (Millipore Sigma #MAB1864, RRID:AB_1679330, 1:1,000), mouse monoclonal GAPDH (Millipore Sigma #MAB374, RRID:AB_2107445, 1:1,000), monoclonal rat α-tubulin (YOL1/34) (Invitrogen #MA1-80189, RRID:AB_2210200, 1:1,000) and monoclonal rabbit HDAC6 (D2E5) antibody (Cell Signaling #7558s, RRID:AB_10891804, 1:1,000) were used.

Techniques: Shear, Immunofluorescence, Control, MANN-WHITNEY, Expressing, Western Blot

Journal: Frontiers in Physiology

Article Title: Endothelial cell elongation and alignment in response to shear stress requires acetylation of microtubules

doi: 10.3389/fphys.2024.1425620

Figure Lengend Snippet: HDAC6 and aTAT1 are key mediators of EC mechanotransduction through regulation of microtubule acetylation. (A) In response to shear stress, endothelial cells elongate and align in the direction of flow through remodeling of the cytoskeleton which includes reorganization of microtubules. There is a flow-dependent increase in acetylation that corresponds to endothelial cell elongation and alignment. HDAC6 and αTAT1 regulate the timing (kinetics) and localization of acetylation on α-tubulin in response to shear stress in endothelial cells. HDAC6 is found at its lowest at the onset of flow. (B) Disruption of HDAC6 and αTAT1, the key regulators of MT acetylation by pharmacological inhibition or through genetic inactivation results in impairment of endothelial cell mechanotransduction. Loss of HDAC6 leads to significant acetylation and faster kinetics in elongation. Inactivation of αTAT1 leads to significant reduction in acetylation with reduced elongation.

Article Snippet: Primary antibodies monoclonal rabbit Acetylated -α-tubulin (Lys40) (D20G3) (Cell Signaling #5335, RRID:AB_10544694, 1:1,000), monoclonal mouse anti-Acetylated α-tubulin (Millipore Sigma #T7451, RRID:AB_609894, 1:1,000), monoclonal mouse anti-Acetylated (Lys40) (6-11B-1) α-tubulin (Cell Signaling #12152s, RRID:AB_2797830, 1:1,000), monoclonal mouse DM1a (Cell Signaling #3873s, RRID:AB_1904178, 1:1,000), polyclonal rabbit anti-detyrosinated tubulin (Millipore Sigma #AB3201, RRID:AB_117350 1:1,000), monoclonal mouse anti-polyglutamylated tubulin, clone B3 (Sigma-Aldrich #T9822,RRID:AB_477598, 1:1,000), monoclonal rat Anti-Tubulin Antibody, clone YL1/2 (Tyrosinated-tubulin) (Millipore Sigma #MAB1864, RRID:AB_1679330, 1:1,000), mouse monoclonal GAPDH (Millipore Sigma #MAB374, RRID:AB_2107445, 1:1,000), monoclonal rat α-tubulin (YOL1/34) (Invitrogen #MA1-80189, RRID:AB_2210200, 1:1,000) and monoclonal rabbit HDAC6 (D2E5) antibody (Cell Signaling #7558s, RRID:AB_10891804, 1:1,000) were used.

Techniques: Shear, Disruption, Inhibition