- PA5-22285 - Invitrogen Antibodies NUMA1 antibody

Gisler S, Maia ARR, Chandrasekaran G, Kopparam J, van Lohuizen M
PloS one 2020;15(4):e0227592

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of NuMA using A) 30 µg HeLa whole cell lysate and B) 30 µg A375 whole cell lysate. Samples were loaded onto a 5% SDS-PAGE gel and probed with a NuMA polyclonal antibody (Product # PA5-22285) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot analysis of NuMA was performed by separating 30 µg of untreated (–) and treated (+) HeLa whole cell extracts by 5% SDS-PAGE. Proteins were transferred to a membrane and probed with a NuMA Polyclonal Antibody (Product # PA5-22285) at a dilution of 1:8000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-NuMA Polyclonal Antibody (Product # PA5-22285) and a 240kDa band corresponding to NuMA was observed in all tested cell lines. Modified whole cell lysates (1% SDS) (30ug lysate) of HeLa (Lane 1), U-2 OS (Lane 2), MCF7 (Lane 3), Caco-2 (Lane 4) and A-431 (Lane 5) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX). 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, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Confocal immunofluorescence analysis (Olympus FV10i) of paraformaldehyde-fixed U2OS, using NuMA antibody (Product # PA5-22285) (Geen) at 1:500 dilution. Alpha-tubulin filaments were labeled with (Product # MA1-25054) (Red) at 1:2,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of NuMA was performed using HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with NuMA Polyclonal Antibody (Product # PA5-22285) at 5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of paraffin-embedded Cal27 Xenograft, using NuMA (Product # PA5-22285) antibody at 1:100 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NuMA Polyclonal Antibody detects NuMA protein at nucleus in human colon carcinoma by immunohistochemical analysis. Sample: Paraffin-embedded human colon carcinoma. NuMA Polyclonal Antibody (Product # PA5-22285) diluted at 1:500. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NuMA Polyclonal Antibody detects NuMA protein at nucleus in human esophageal cancer by immunohistochemical analysis. Sample: Paraffin-embedded human esophageal cancer. NuMA Polyclonal Antibody (Product # PA5-22285) diluted at 1:500. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 3 Cas9-induced NUMA1 knockout rescues HAP1 cytotoxicity caused by BMI1 inhibition. (A) Protein expression of NUMA1 in populations of HAP1 cells transduced with a doxycycline-inducible shBMI1 construct in NUMA1-WT or NUMA1-KO backgrounds (NUMA1-KO1 and NUMA1-KO2 represent two separate clones). (B and C) Survival of the HAP1 NUMA1-WT and NUMA1-KO clones transduced with the inducible shBMI1 construct and maintained with or without doxycycline (B) colony formation and (C) cell viability. (D) Expression of NUMA1 protein in HAP1 NUMA1-WT and NUMA1-KO clones treated either with 0.1% DMSO or 40 nM of PTC-318. The survival of these cells was analyzed by (E) colony formation assay and (F) cell viability. (G) Protein expression of NUMA1 and BMI1 in NUMA1-WT cells transfected with CRISPR-Cas9 targeting BMI1, single-cell sorted, and expanded (left). (H) Percentages of each genetic background based on the respective protein expression (right). Error bars represent SD (n = 3). A two-tailed Student''s t-test was performed for statistical testing.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 5 Non-small cell lung cancer cell lines are sensitive to BMI1 inhibition. (A) IC50 measurements of NSCLC cell lines h520, SK-MES-1, and SWI-1573. The x-axis shows the PTC-318 concentration in muM and the y-axis the percentage of surviving cells relative to non-treated control counterparts. (B) Colony formation of h520 and SK-MES-1 cells treated with either 0.1% DMSO or 40 nM (h520) or 80 nM (SK-MES-1) of PTC-318. (C) Percentages of h520 phospho-histone H3 populations after treatment with either 0.1% DMSO or 40 nM PTC-318 determined by flow cytometry quantification. (D) Percentages of h520 phospho-histone H3 populations after treatment with either 0.1% DMSO or 80 nM PTC-318 determined by flow cytometry quantification. Error bars represent SD (n = 3). A two-tailed Student''s t-test was performed for statistical testing. (E) Protein expression of NUMA1 and BMI1 in NUMA1-WT clones of an h520 population transfected with Cas9 targeting BMI1, single-cell sorted, and expanded.

PA5-22285