- PA3-16832 - Invitrogen Antibodies TPX2 antibody

Fang CT, Kuo HH, Hsu SC, Yih LH
Cell death & disease 2020 Sep 1;11(8):715

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-TPX2 Polyclonal Antibody (Product # PA3-16832) and a ~110 kDa band corresponding to targeting protein was observed along with an uncharacterised band (*) at ~40 kDa across cell lines tested. Nuclear enriched extracts (40 µg lysate) of HeLa (Lane 1), A431 (Lane 2), A549 (Lane 3), MCF7(Lane 4), HCT116 (Lane 5) and NIH/3T3 (Lane 6) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:20000 dilution) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of TPX2 in 1) HeLa, 2) Ntera2, 3) K-562 and 4) Raji whole cell lysates. Samples were incubated in TPX2 polyclonal antibody (Product # PA3-16832).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of Targeting protein for Xklp2 was achieved by transfecting HeLa with Targeting protein for Xklp2 specific siRNAs (Silencer® select Product # S22745, S22746). Western blot analysis (Fig. a) was performed using Nuclear enriched extracts from the Targeting protein for Xklp2 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with TPX2 Polyclonal Antibody (Product # PA3-16832, 1:1000 dilution ) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20000 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 Targeting protein for Xklp2.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of TPX2 in HeLa cell lysate. Samples were incubated in TPX2 polyclonal (Product # PA3-16832) using a dilution of 1:250. Electropherogram image of corresponding Simple Western lane view. Image reported by internal validation.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemistry analysis of TPX2 in HeLa cells fixed in 3.5% paraformaldehyde. Samples were incubated in TPX2 polyclonal antibody (Product # PA3-16832) using a dilution of 1:1000. Nuclear staining during interphase and spindle staining during mitosis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 5 HSP70 is required for proper Eg5 interactions with TPX2, acetylated tubulin and alpha-tubulin. Representative images of Eg5-TPX2 PLA ( a ), Eg5-alpha-tubulin PLA ( c ) and Eg5-acetyl-tubulin PLA ( e ) signals are shown in red in untreated and PES- or VER-treated prometaphase and metaphase cells counterstained with beta-tubulin (green) and DAPI (blue). The ratio of the PLA signal at the poles to the whole-cell signal was measured as described, and the interquartile distributions from two independent experiments are shown for ( b ) Eg5-TPX2, ( d ) Eg5-alpha-tubulin, and ( f ) Eg5-acetyl-tubulin. The numbers in parentheses indicate the number of the cells measured. * p < 0.05 compared to untreated by Mann-Whitney Rank Sum test. g (upper) BMH-mediated crosslinking was performed as described, revealing a protein complex >460 kD containing Eg5 and TPX2 in synchronized mitotic cells that were untreated or treated with PES or VER. (Lower) Total Eg5 and TPX2 proteins in the denatured lysate. h The bar charts show the levels of Eg5 and TPX2 compared to the untreated cells, as normalized by the mitosis marker p-histone3 for BMH-crosslinked samples (upper) and no BMH-treated samples (lower). Mean +- SD of six replicates from three independent experiments is shown. * p < 0.05 compared to untreated by Student's t test.

PA3-16832