32-2700

antibody from Invitrogen Antibodies

Targeting: TUBA1C bcm948, MGC10851, MGC14580, TUBA6

Provider product page for 32-2700

Western blot

Immunocytochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [54]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [1]
Flow cytometry [1]
Other assay [32]

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Product number: 32-2700 - Provider product page
Provider: Invitrogen Antibodies
Product name: Acetyl-alpha Tubulin (Lys40) Monoclonal Antibody (6-11B-1)
Antibody type: Monoclonal
Antigen: Other
Description: This antibody reacts with the ~ 55 kDa acetylated form of alpha-tubulin. This antibody reacts with human, mouse, and rat. Reactivity is confirmed with NIH3T3 and HeLa cells.
Reactivity: Human, Mouse, Rat
Host: Mouse
Isotype: IgG
Antibody clone number: 6-11B-1
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: -20°C

TUBA1C protein structure - 32-2700 shown in red.

Supportive validation

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Experimental details: Fig. 4 a HDAC8 interacts and deacetylates alpha tubulin. a HDAC8 and Acetylated Alpha tubulin Interaction in HeLa and HEK 293 T cells as demonstrated by immunoprecipitation using HDAC8 antibody followed by immunoblot analysis using ac-alpha tubulin. b ac-alpha tubulin levels increase when HDAC8 is inhibited with PCI-34051 (20 muM) or when treated with PKA activator, Forskolin (10 muM) and in HeLa and HEK 293 T cells. c Densitometry analysis of the immunoblot bands showing fold change in expression pattern of HDAC8, Alpha tubulin and acetylated tubulin normalized with GAPDH. d Lower panel of chromatogram represents for Unac-alpha tubulin peptide with a retention time of 14.70 Min. Middle panel of chromatogram for ac-alpha tubulin with retention time of 15.37 Min. Upper panel of chromatogram for GST-HDAC8 (30 mug) + ac-alpha tubulin (500 nM) with a retention time of 14.72 Min. Shift in the retention time from a (15.37) to b (14.72) clearly demonstrates the in vitro deacetylation of ac-alpha tubulin by GST-HDAC8

Supportive validation

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Supportive validation

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Supportive validation

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Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 4 a HDAC8 interacts and deacetylates alpha tubulin. a HDAC8 and Acetylated Alpha tubulin Interaction in HeLa and HEK 293 T cells as demonstrated by immunoprecipitation using HDAC8 antibody followed by immunoblot analysis using ac-alpha tubulin. b ac-alpha tubulin levels increase when HDAC8 is inhibited with PCI-34051 (20 muM) or when treated with PKA activator, Forskolin (10 muM) and in HeLa and HEK 293 T cells. c Densitometry analysis of the immunoblot bands showing fold change in expression pattern of HDAC8, Alpha tubulin and acetylated tubulin normalized with GAPDH. d Lower panel of chromatogram represents for Unac-alpha tubulin peptide with a retention time of 14.70 Min. Middle panel of chromatogram for ac-alpha tubulin with retention time of 15.37 Min. Upper panel of chromatogram for GST-HDAC8 (30 mug) + ac-alpha tubulin (500 nM) with a retention time of 14.72 Min. Shift in the retention time from a (15.37) to b (14.72) clearly demonstrates the in vitro deacetylation of ac-alpha tubulin by GST-HDAC8

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 5 HDAC8 might be primary tubulin deacetylase in HeLa cells. a Immunoblot showing the protein expression levels of HDAC8 and HDAC6 in various cancer cell lines. b Densitometric analysis of the (a) HDAC6 bands and (b) HDAC8 bands of immuoblot A. c Real time analysis confirms higher HDAC8 expression compared to HDAC6 in HeLa and HEK 293T normalized with GAPDH * indicates p-value

Supportive validation

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Experimental details: 1 Supplementary Figure 1. Cilia quantification in MEFs a . Staining for cilia (acetylated tubulin, red, arrow) in serum starved MEFs with reapplication of serum. These results are quantified at the right for at least 200 cells. *** P

Supportive validation

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Experimental details: 5 Supplementary Figure 5. Dnchc2 siRNA and aphidicolin treatment effect on ciliation a . Luciferase activity in MEFs without serum starvation and transfected with 5 pmol Dnchc2 siRNA and treated with WCM or LCM. N.S.=not significant, n=3 independent experiments, Student's t -test. Error bars represent S.E.M. b . Staining for cilia (acetylated tubulin, red, arrow) in MEFs transfected with low dose and high dose (3-fold higher) Dnchc2 siRNA. Hoechst (blue) labels nuclei. These results are quantified in the panel on the right for at least 200 cells. *** P

Supportive validation

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Experimental details: Figure 1 The primary cilium dampens Wnt activity by regulating beta-catenin a . Luciferase response to Wnt3a conditioned media in a wild-type MEF cell line following serum starvation and re-addition. * P

Supportive validation

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Experimental details: Figure 3 The primary cilium sequesters Jouberin and beta-catenin away from the nucleus a . 3T3s transfected with two siRNAs to Dnchc2 exhibit accumulation of Jbn-GFP (green) along the length of the cilium (labeled with antibody to acetylated tubulin, red). The nucleus is labeled with Hoechst (blue). Schematic represents the experimental approach and interpretation of these results. Quantification of the green fluorescence along the cilium reveals a shifted localization along the ciliary axoneme in siRNA transfected cells with significantly increased levels of GFP beyond the basal body. Average fluorescence intensities of cells transfected with negative control siRNA (n=7), Dnchc2 siRNA #1 (n=10), or Dnchc2 siRNA #2 (n=11) were normalized and displayed in arbitrary grey value units. Position along the cilium was normalized for total cilia length and results were binned into 20 equal segments. b . Luciferase activity in MEFs transfected with 5 pmol of Dnchc2 siRNA and treated with Wnt3a conditioned media (WCM) or control L-cell conditioned media (LCM). * P

Supportive validation

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Experimental details: Figure 5 Regulation of cancer cell proliferation by cilia-localized Jouberin a . BrdU staining (red) in MEFs transfected with GFP tagged wild-type or DeltaRVxP mutant construct (green). Hoechst labels nuclei (blue). Arrows point to GFP positive transfected cells. Quantification of the percent of 200 counted GFP positive cells which stained positive for BrdU is shown at right. ** P

Supportive validation

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Experimental details: 2 Supplementary Figure 2. Kif3a knockdown and quantification of ciliation a . Western blot analysis of whole cell lysates from 293Ts transfected with Kif3A siRNA. Alpha-tubulin is shown as a loading control. b . Staining for cilia (acetylated tubulin, red) in MEFs transfected with Kif3a siRNA #3. Arrows point to cilia in control transfected cells. Hoechst (blue) labels nuclei. Results are quantified at the right from at least 200 cells for each. *** P

Supportive validation

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Experimental details: 3 Supplementary Figure 3. beta-catenin and Jbn are sequestered in the presence of cilia a . Staining for endogenous beta-catenin (green) in ciliated (actylated tubulin, red) and nonciliated (arrow) IMCDs revealing increased nuclear (Hoechst, blue, outlined) localization of beta-catenin in nonciliated cells compared with ciliated neighboring cells. b . Quantification of nuclear beta-catenin staining in ciliated and nonciliated IMCDs. Mean grey values were calculated using ImageJ software (NIH) to create a binary mask from Hoechst staining as a nuclear boundary for measurement of beta-catenin levels. * P

Supportive validation

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Experimental details: 4 Supplementary Figure 4. Loss of cilia potentiates Jbn's role in the Wnt pathway a . 293T cells stained for cilia with antibody to acetylated tubulin (red) display clear cilia with Jbn-GFP (green) localized to the basal body similar to previously described in other ciliated cells 2 , 38 . Hoechst was used to stain the nucleus (blue). b . Cilia staining of 293T cells with two independent markers: acetylated tubulin and Arl13b. Arl13b reveals a greater number of cilia (44% of cells) than acetylated tubulin staining (10% of cells). c . Luciferase activity in MEFs transfected with Kif3a siRNA and overexpressed Jbn or EV with activation of the pathway with cotransfected beta-catDeltaN. * P

Supportive validation

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Experimental details: Figure 8 ATRA increased the number of ciliated cells in HT and LT. ( A ) Immunofluorescence of omentum-derived mesothelial cells (control) and effluent-derived mesothelial cells from LT and HT grown until confluence in the presence of ATRA (0, 50, 100 and 200 nM). Cells were labeled with anti-acetylated alpha-tubulin (green). Nuclei were labeled with ToPro-3 (blue). HT and LT cultures exhibited a reduction in the number of ciliated cells (e and i, respectively). ATRA increased the number of ciliated cells in LT (f, g and h) and HT (j, k and l), being more notable in HT. In control, ATRA decreased the number of ciliated cells. Panel k shows the zoom of a cell with two cilia. (B to E) Quantification of number of ciliated cells. Mean +- SEM of three individual experiments from three different patients are shown. ( B ) * P

Supportive validation

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Experimental details: Fig. 3 HDAC 1 and HDAC 6 increase MMP-2/9 activity and decrease acetylated alpha-tubulin levels, respectively. a ) C2 and 786-0 parental and knocked down HDAC 1 were analyzed for MMP activity by gelatin zymography assay. MMP activity was measured both in cell lysates as well as cell supernatants. b ) Renal tumor cell lines were analyzed for HDAC 6 and acetylated alpha-tubulin expression by immunofluorescence. The staining in red indicates acetylated alpha-tubulin and the staining in green indicates HDAC 6 expression. Scale bar indicates 50 muM distance and images are taken at 20X magnification. c ) Image J analysis measured immunofluorescence by calculating integrated density values of at least three representative fields per cell line. * p < 0.05 indicates statistically significant difference in acetylated alpha-tubulin levels as compared to C2 cells. The error bars represent standard errors from triplicate experiments and p-value was calculated using students t -test

Supportive validation

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Experimental details: Figure 5 Data implicating Sept8/ SEPTIN8 in the human tubule hypoxia cell culture model. ( A ) RNAseq values of genes within rat LD block within the RPTEC TERT1 normoxic vs hypoxic conditions. Genes on the right are significant genes. ( B ) TPM values in each of the RNAseq groups for SEPTIN8 in the RPTEC TERT1 cells. ( C ) Expression of SEPTIN8 in single cell datasets (human and mouse). ( D ) Immunohistochemistry from the Human Protein Altas (modified from www.proteinatlas.org/ENSG00000164402-SEPT8/tissue/kidney ) for SEPTIN8 using HPA005665 antibody. Labels are added for tubules and glomerulus. ( E ) Immunofluorescence of SEPTIN8 (red) with acetyl-alpha tubulin, actin, or ZO-1 (green) as labeled. SEPTIN8 localizes with acetyl-alpha tubulin under shear stress with normoxic conditions. ( F ) Under hypoxic conditions, however, SEPTIN8 re-localizes near the actin filaments.

Supportive validation

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Experimental details: Figure EV3 Tubulin acetylation is increased by tubacin, but not by the Aurora A inhibitor WT neurons (DIV12) were treated with tubacin (1 muM) or TC-S 7010 (7 nM) for 48 h or left untreated. Acetylated alpha-tubulin (ac-Tub) was measured by Western blot in untreated and tubacin or TC-S-treated cells. Representative bands of acetylated alpha-tubulin are depicted above the histogram, which shows the mean +- SE of the percentage of the expression levels compared to untreated samples ( n = 3, * P < 0.05, one-way ANOVA followed by Dunn's post hoc test). Data are normalized to total protein content, visualized by a TGX stain-free technology.

Supportive validation

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Experimental details: FIGURE 9 SHH and acetylated alpha-tubulin protein localization in the rostral palatal shelves in pre-hatching chameleons. Immunodetection of SHH (red) and acetylated alpha-tubulin (green) proteins on transversal sections. Lower power pictures overview localization at 77 dpo (A) , 98 dpo (B) , and 105 dpo (C) during pre-hatching development. White rectangles define regions focused on ventral (v), middle (m) and dorsal (d) parts of the palatal shelves. Pictures (A v -C d ) show higher power details from ventral, middle and dorsal regions. White arrowheads indicate polarized colocalization of SHH and primary cilium (detected using acetylated alpha-tubulin) on the same cellular side. Acetylated alpha-tubulin is present not only in primary cilia, but as well in microtubules of mitotic spindle, therefore there was signal in both structures detected in green. Nuclei are counterstained with DRAQ5. Scale bar: 20 mum.

Supportive validation

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Experimental details: Figure 2 Prolonged cilia contacts form in cultured cells . ( A ) MDCK cells were cultured in a matrix to form a three-dimensional cyst, then fixed and stained. The arrow indicates an area of overlapping contact (acetylated tubulin - green; Hoechst - blue; actin - red). ( B , C ) MDCK cells were grown on transwell filters, fixed, stained, and imaged using a confocal microscope. These maximum intensity projection images show overlapping ( B ) and point ( C ) contacts between cilia (acetylated tubulin - green; Hoechst - blue; ZO1 - red). ( D) Filter grown MDCK cells were stained and imaged on a confocal microscope (Hoechst - blue, anti-detyrosinated tubulin - green, and anti-gamma tubulin - red). The image presented is a maximum intensity projection (the contrast in the red channel was changed to make the centrosomes more visible). ( E , F ) Filter grown MDCK cells stably expressing tubulin-GFP were imaged by confocal microscopy and used to generate projection images. Cilia from nearby cells appear to be coming together. ( G ) We monitored cells during contact formation and measured how long cilia remained together to determine whether the contacts are transient chance encounters or more persistent inter-cellular interactions. Each point on the graph represents a contact event. The shortest contact duration observed was just over 2 h. ( H ) To test whether primary cilia can move we collected rapid z stacks of primary cilia from TubGFP expressing cells at 4.5 s intervals using a

Supportive validation

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Experimental details: Fig. 5 Acetylated alpha-tubulin is regulated by HDAC6/ER-alpha interaction in RCC cell lines. a ) HDAC 6, ER-alpha and acetylated alpha-tubulin protein expression were measured by western blot analysis in ccRCC tumors and adjacent non-tumor tissue. b ) A representative ccRCC tumor showed HDAC 6 (in red) and ER-alpha (in green) localization in the cytoplasm. c - d ) Representative immunofluorescent images of acetylated alpha-tubulin (in red) and HDAC 6 (in green) expression in parental and ER-alpha knockdown cell lines are shown. e ) Knockdown of ER-alpha in MCF-7 and renal cell tumors as measured by Western blot analysis. f ) Image J analysis measured immunofluorescence by calculating integrated density values of at least three representative fields per cell line. * p < 0.05 and ** p < 0.01 indicates statistically significant difference of acetylated alpha-tubulin levels in ER-alpha knockdown cells as compared to the parental cell lines. The error bars represent standard errors from triplicate experiments and p-value was calculated using students t -test. g ) C2H6 cells treatment with 10 muM hydroxy tamoxifen and/or 50nM panobinostat for 4 h. Representative immunofluorescence images of acetylated alpha-tubulin (in red), HDAC 6 (in green) and ER-alpha (in green) are shown. h ) Image J analysis measured immunofluorescence by calculating integrated density values of at least three representative fields per cell line. *p < 0.01 indicates statistically different HDAC 6 l

Supportive validation

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Experimental details: Figure 2 Localization of Select Centriolar Components (A-C) SIM images of purified Chlamydomonas centrioles stained with acetylated tubulin (green, all panels), highlighting the proximal localization of the cartwheel components CrSAS-6 (A, red), Bld10p (B, blue), and the central core localization of monoglutamylated tubulin (MonoE; C, dark blue). The scale bars represent 100 nm. (D, F, and H) Superimposed plot profiles of acetylated tubulin (green) with CrSAS-6 (red) (D), Bld10p (blue) (F), or monoglutamylated tubulin (dark blue) (MonoE) (H). (E, G, and I) Distribution of CrSAS-6 (E), Bld10p (G), and MonoE (I) as compared to that of acetylated tubulin (left), together with corresponding schematic representations of centriole (right). Heights were as follows: CrSAS-6: 123 +- 20 nm; Bld10p: 133 +- 33 nm; MonoE: 286 +- 33 nm. Error bars represent SD. See also Figure S1 .

Supportive validation

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Experimental details: Figure 4 POB15 Is a Chlamydomonas Central Core Protein (A) Schematic representation of the 820-amino-acid-long POB15 protein highlighting its predicted BTB/POZ and coiled coil (CC) domains. (B-D) Immunofluorescence microscopy of Chlamydomonas control (B), bld10 mutant (C), and bld12 mutant (D) cells stained with antibodies against acetylated tubulin (green) and POB15 (magenta). Here and in other figures, insets are 2-fold magnified views of the region indicated in the lower magnification views, with dashed contours indicating those mutant cases in which centrioles are lacking, shown for visualization purposes, as it is not possible to ascertain where exactly below the plasma membrane centrioles would have been. The scale bar represents 1 mum. (E) SIM image of purified Chlamydomonas centrioles viewed from the side and stained with antibodies against acetylated tubulin (green) and POB15 (magenta). Note the central core localization of the bulk of POB15, as well as the proximal focus (arrowhead). The scale bars represent 100 nm. (F) Superimposed plot profiles of acetylated tubulin (green) and POB15 (magenta). (G) Schematic representation of POB15 localization (height: 229 +- 29 nm; magenta) as compared to that of acetylated tubulin (green). Data are represented as mean +- SD. (H and I) SIM images of centrioles stained with MonoE (green) and POB15 (magenta). The scale bars represent 100 nm. (J) Top view of centrioles imaged using dual SIM/STORM microscopy. Ima

Supportive validation

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Experimental details: EV1 Figure GRK 2 is not required for ciliogenesis or IFT A-C Primary cilia are normal in GRK2 -/- fibroblasts. Cells were serum starved for 24 or 48 h to induce cilia, and (A) immunostained for acetylated tubulin (AcTu, red), ARL13B (green) (upper panel; insets show the individual signals), or AcTu (red), pericentrin (red) and GLI3 (green) (lower panel). Control and GRK2 -/- cilia were positive for AcTu and ARL13B, and showed similar localization of GLI3 to the tips (lower panel, arrows). Acetylated tubulin and pericentrin staining were used to visualize the axoneme and centrioles, respectively. Scale bar, 2 mum. (B) Length distribution of cilia did not differ between control and GRK2 -/- cells. Red bars show medians; dots represent individual cilia. Mann-Whitney U test; number of biological experiments and the total numbers of analyzed cilia are indicated. (C) There was no difference in the efficiency of ciliogenesis between control and GRK2 -/- cells. Mean +- SEM. Welch's t -test; number of biological experiments and the total numbers of analyzed cells are indicated. D Loss of GRK2 did not affect localization of ciliary and IFT components. Immunofluorescence of serum-starved (48 hr) control and GRK2 -/- fibroblasts immunostained with AcTu or detyrosinated tubulin (DetyrTu) to mark the cilium (red), and IFT43, IFT88, KIF3A, TRAF3IP1, WDR34, or ICK (green). No significant differences in staining were found between control and GRK2 -/- cilia, suggesting normal IFT. Scale bars,

Supportive validation

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Experimental details: Fig. 3 Conditional deletion of Nedd4-2 in lung epithelial cells causes peripheral airway remodeling with increased Muc5b production. a - d Representative micrographs and high magnification insets illustrating the airway cell population of terminal bronchioles from conditional Nedd4-2 -/- mice and littermate controls after 3 months of doxycycline induction stained with Alcian blue and periodic acid-Schiff (AB-PAS) (control, n = 5 mice; Nedd4-2 -/- , n = 9 mice) ( a ), or co-stained with anti-Muc5b and anti-acetylated alpha-tubulin antibodies (control, n = 5 mice; Nedd4-2 -/- , n = 6) ( b , c ), or with anti-Muc5ac and anti-CCSP antibodies ( n = 4/group) ( d ). Scale bars, 60 and 15 um (insets). e - i Summary of numeric densities of club cells ( n = 5 mice/group) ( e ), ciliated cells ( n = 6/group and proximal, conditional Nedd4-2 -/- , n = 5 mice) ( f ), goblet cells (proximal, control, n = 5 mice; conditional Nedd4-2 -/- , n = 6 mice; distal, control, n = 5 mice; conditional Nedd4-2 -/- , n = 7 mice, terminal, n = 9/group) ( g ), Muc5b expressing cells (proximal, n = 5/group; distal, control, n = 5 mice; conditional Nedd4-2 -/- , n = 6 mice, terminal, n = 6/group) ( h ), and Muc5ac expressing cells ( n = 4/group) ( i ) in proximal, distal and terminal conducting airways of conditional Nedd4-2 -/- mice and littermate controls after 3 months of doxycycline induction (BM basement membrane). j , k mRNA expression levels of Muc5b ( j ) and Muc5ac (control, n = 15 mice; conditiona