- MA1-19421 - Invitrogen Antibodies TUBG1 antibody

Submitted references Pathogenic LRRK2 regulates ciliation probability upstream of tau tubulin kinase 2 via Rab10 and RILPL1 proteins.

Sobu Y, Wawro PS, Dhekne HS, Yeshaw WM, Pfeffer SR
Proceedings of the National Academy of Sciences of the United States of America 2021 Mar 9;118(10)

WDR62 regulates spindle dynamics as an adaptor protein between TPX2/Aurora A and katanin.

Huang J, Liang Z, Guan C, Hua S, Jiang K
The Journal of cell biology 2021 Aug 2;220(8)

Loss of AMPKalpha1 Triggers Centrosome Amplification via PLK4 Upregulation in Mouse Embryonic Fibroblasts.

Zhao Q, Coughlan KA, Zou MH, Song P
International journal of molecular sciences 2020 Apr 16;21(8)

Drp1 Controls Effective T Cell Immune-Surveillance by Regulating T Cell Migration, Proliferation, and cMyc-Dependent Metabolic Reprogramming.

Simula L, Pacella I, Colamatteo A, Procaccini C, Cancila V, Bordi M, Tregnago C, Corrado M, Pigazzi M, Barnaba V, Tripodo C, Matarese G, Piconese S, Campello S
Cell reports 2018 Dec 11;25(11):3059-3073.e10

Plk1-dependent recruitment of gamma-tubulin complexes to mitotic centrosomes involves multiple PCM components.

Haren L, Stearns T, Lüders J
PloS one 2009 Jun 19;4(6):e5976

Nuclear gamma-tubulin during acentriolar plant mitosis.

Binarová P, Cenklová V, Hause B, Kubátová E, Lysák M, Dolezel J, Bögre L, Dráber P
The Plant cell 2000 Mar;12(3):433-42

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blotting analysis of human gamma-tubulin using mouse monoclonal antibody TU-30 on lysates of various cell lines under reducing and non-reducing conditions. Nitrocellulose membrane was probed with 2µg/mL of mouse anti-gamma-tubulin monoclonal antibody (Product # MA1-19421) followed by IRDye800-conjugated anti-mouse secondary antibody. A specific band was detected for gamma-tubulin at approximately 46kDa.

Supportive validation

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Experimental details: Western blotting analysis of differential reactivity of monoclonal antibodies to γ-tubulin with human γ-tubulin isotypes. (A) Immunoblots of total cell lysates from SH-SY5Y cells, expressing TagRFP-tagged human γ-tubulin 1 (γ-Tb1) or γ-tubulin 2 (γ-Tb2), probed with antibodies to γ-tubulin (TU-30) (Product # MA1-19421), γ-tubulin (TU-32), TagRFP (RFP) and GAPDH. In control samples, only secondary anti-mouse antibody was applied. (B) Immunoblots of immobilized GST-tagged human C-terminal regions (a.a. 362-451) of γ-Tb1 or γ-Tb2 probed with antibodies to γ-tubulin (TU-30, TU-32) and GST. In control samples, only secondary anti-mouse antibody was applied.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of gamma Tubulin was achieved by transfecting HeLa cells with gamma Tubulin specific siRNAs (Silencer® select Product # s14502). Western blot analysis (Fig. a) was performed using membrane extracts from the gamma Tubulin knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with gamma Tubulin Monoclonal Antibody (TU30) (Product # MA1-19421, 1:1000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to gamma Tubulin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of HeLa (Lane 1), MCF7 (Lane 2), A549 (Lane 3), Hep G2 (Lane 4), Caco-2 (Lane 5), A-431 (Lane 6), C2C12 (Lane 7), NIH/3T3 (Lane 8) and SH-SY5Y (Lane 9). The blot was probed with Anti-gamma Tubulin Monoclonal Antibody (TU30) (Product # MA1-19421, 1:1000 dilution) and detected by chemiluminescence using Goat anti Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). A 51 kDa band corresponding to gamma Tubulin was observed across all cell lines tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of gamma Tubulin using a monoclonal antibody (Product # MA1-19421).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemistry staining of microtubular networks in 3T3 mouse fibroblasts. A – metaphase; B – anaphase; C - telophase. Gamma-tubulin (red) stained with anti-gamma-tubulin (TU-30) Monoclonal antibody (Product # MA1-19421), alpha-tubulin (green) with polyclonal anti-alpha-tubulin antibody and nuclei with DAPI (blue).

Supportive validation

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Experimental details: Immunocytochemistry staining of murine fibroblasts using anti-gamma-tubulin (TU-30; direct conjugate with Dyomics 547, red) Monoclonal antibody (Product # MA1-19421). Nuclei were stained with DAPI (blue).

Supportive validation

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Experimental details: Immunocytochemistry staining of P19X1 mouse embryonal carcinoma cell line using anti-gamma-tubulin (TU-30) Monoclonal antibody (Product # MA1-19421), detection by secondary antibody Goat anti-mouse Cy3. Nuclei were stained with DAPI (blue).

Supportive validation

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Experimental details: Separation of MCF-7 cells stained using anti-gamma-Tubulin (TU-30) purified Monoclonal antibody (Product # MA1-19421) (concentration in sample 9 µg/mL, GAM APC, red-filled) from MCF-7 cells unstained by primary antibody (GAM APC, black-dashed) in flow cytometry analysis (intracellular staining).

Supportive validation

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Experimental details: Figure 2 AMPKalpha1 deletion confers centrosome amplification in MEFs. ( a ) Representative images of centrosome morphologies of WT and AMPKalpha1 -/- MEFs (scale bar = 20 mum) in the prometaphase of the cell cycle are shown. Centrosomes, spindles, and DNA were co-stained with anti-gamma-tubulin antibody (red), anti-alpha-tubulin antibody (green), and 4.6-diamidino-2-phenylindole (DAPI, blue), respectively, and visualized by fluorescence microscope; ( b ) Quantification data for the percentage of MEFs containing >2 centrosomes are presented. n = 5, * p < 0.01, AMPKalpha1 -/- vs. WT; ( c ) Representative images of centrosome morphologies of WT and AMPKalpha1 -/- MEFs (scale bar = 20 mum) in the metaphase of the cell cycle are shown; ( d ) Percentage of cells with >2 centrosomes. n = 5, * p < 0.01, AMPKalpha1 -/- vs. WT.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 PLK4 elevation is p52-mediated. ( a ) p52 knockdown blocked the PLK4 mRNA upregulation in AMPKalpha1 -/- MEFs. n = 3, * p < 0.01, vs. C siRNA/WT; + p < 0.01, vs. C siRNA/AMPKalpha1 -/- ; ( b ) Representative Western blotting showed p52 siRNA decreased the PLK4 protein elevated in AMPKalpha1 -/- MEFs. STE: short-term exposure; LTE: long-term exposure; ( c ) Quantification data of PLK4 protein levels for (b); n = 4, * p < 0.01, vs. C siRNA/WT; + p < 0.01, vs. C siRNA/AMPKalpha1 -/- ; ( d ) MEFs were treated with p52 siRNA or control siRNA for 72 h before the start of the experiment. Cells were treated with 20 ng/mL nocodazole for 4 hours to depolymerize microtubules. Centrosomes, spindles, and DNA were co-stained with anti-gamma-tubulin antibody (red), anti-alpha-tubulin antibody (green), and 4.6-diamidino-2-phenylindole (DAPI, blue), respectively, and visualized by fluorescence microscope (scale bar = 20 mum). Representative images are shown; ( e ) Quantification data represent the mean +- S.D. from three separate experiments. * p < 0.01, vs. WT/control siRNA; + p < 0.01, vs. AMPKalpha1 -/- /control siRNA. C siRNA: control siRNA.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 PLK4 inhibition reverses centrosome amplification and cell hyperproliferation in AMPKalpha1-deleted MEFs. ( a ) PLK4 inhibition by Centrinone blocked centrosome amplification in AMPKalpha1 -/- MEFs. MEFs were treated with Centrinone (125 nM) or vehicle (DMSO) for 16 h. The cells were treated with nocodazole (20 ng/mL) for 4 hours to depolymerize the microtubules. Then, centrosomes, spindles, and DNA were co-stained with anti-gamma-tubulin antibody (red), anti-alpha-tubulin antibody (green), and 4.6-diamidino-2-phenylindole (DAPI, blue), respectively, and visualized by fluorescence microscope (scale bar = 20 mum); ( b ) PLK4 inhibition restrained cellular hyperproliferation in AMPKalpha1 -/- MEFs. MEF cells were seeded into 10-cm tissue culture dishes at 1 x 10 5 cells/dish. Centrinone was added at 125 nM. Every day, cells were harvested, counted using a Bio-Rad cell counter, and re-seeded into new dishes. Quantification data represent the mean +- S.D. from three separate experiments. * p < 0.01, vs. alpha1 -/- + Centrinone; ( c ) A scheme illustrating the role of p52-PLK4 in AMPKalpha1 deletion-mediated centrosome amplification. P52 transcriptionally regulates PLK4, which contributes to centrosome amplification and the consequent chromosome instability.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 5. Pathogenic LRRK2 activity blocks TTBK2 recruitment to the mother centriole. ( A ) R1441C LRRK2 MEF cells stably expressing GFP-TTBK2 were starved for 24 h +- MLi-2. Cells were then fixed with cold methanol and stained with rabbit anti-TTBK2 (green) and mouse anti-gamma-tubulin (red) antibodies. At right are enlarged regions boxed in the larger images at left. ( B ) Percent of cells with TTBK2 at the centrosome, marked by gamma-tubulin staining. Values represent the mean and SEM of three independent experiments ( n > 60 in each experiment). Significance was determined by the paired t test; * P = 0.004. (Scale bars, 10 um.)

MA1-19421

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