- MA5-14871 - Invitrogen Antibodies ATM antibody

Qi L, Chakravarthy R, Li MM, Deng CX, Li R, Hu Y
iScience 2022 Sep 16;25(9):104944

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of ATM was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR901163_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of ATM was performed by loading 30 µg of HeLa Wild Type (Lane 1), HeLa Cas9 (Lane 2) andHeLa ATM KO (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ 3-8% Tris-Acetate Protein Gel (Product # EA0378BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-ATM Monoclonal Antibody (C.74.4) (Product # MA5-14871, 1:1,000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to ATM.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of Serine-protein kinase ATM was achieved by transfecting Hep G2 with Serine-protein kinase ATM specific siRNAs (Silencer® select Product # s1708, s1709). Western blot analysis (Fig. a) was performed using Whole cell extracts from the Serine-protein kinase ATM knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with ATM Monoclonal Antibody (C.74.4) (Product # MA5-14871, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 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 Serine-protein kinase ATM.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-ATM Monoclonal Antibody (C.74.4) (Product # MA5-14871) and a 350kDa band corresponding to Serine-protein kinase ATM was observed across cell lines tested. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), U-2 OS (Lane 2), Hep G2 (Lane 3), K-562 (Lane 4), Jurkat (Lane 5) and NIH/3T3 (Lane 6) were electrophoresed using NuPAGE™ 3-8% Tris-Acetate Protein Gel (Product # EA0378BOX). 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 Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Abrogation of ATM phosphorylation on BRCA1-S1152 reduces SKP2 recruitment and ATM-NBS1 interaction upon DNA damage (A) Coimmunoprecipitation shows reduced interaction between BRCA1-S1198A and SKP2 in HEK293 cells. (B) Recruitment of SKP2 upon microirradiation. SKP2 laser stripes (green) and gammaH2AX (red) at 10, 30, and 60 min time points are shown. Scale bar, 10 mum. Bar graph below is quantification of three independent experiments. For 10 min (n = 326 for Brca1 WT and n = 351 for Brca1 S1152A/S1152A , p = 0.007656); 30 min (n = 336 for Brca1 WT and n = 427 for Brca1 S1152A/S1152A , p = 0.009519); 60 min (n = 336 for Brca1 WT and n = 380 for Brca1 S1152A/S1152A , p = 0.009258). (C) Immunoprecipitation of endogenous NBS1 shows reduced K63-linked ubiquitination of NBS1 in Brca1 S1152A/S1152A MEF cells, but not in Brca1 S971A/S971A MEF cells, in response to DNA damage. (D) Coimmunoprecipitation shows reduced interaction between NBS1 and ATM upon DNA damage in Brca1 S1152A/S1152A MEF cells but not in Brca1 S971A/S971A MEF cells.

MA5-14871