- 14-5158-82 - Invitrogen Antibodies CGAS antibody

Omer A, Barrera MC, Moran JL, Lian XJ, Di Marco S, Beausejour C, Gallouzi IE
Nature communications 2020 Oct 5;11(1):4979

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot of human cGAS using reduced: 1) Daudi human peripheral blood cell line, 2) HEK-293 human embryonic kidney cell line, 3) HEL human erythroblast cell line, 4) HeLa human cervix epithelial cell line, 5) MCF7 human mammary gland epithelial cell line, 6) NIH/3T3 mouse embryo fibroblast cell line, and 7) THP-1 human myeloid peripheral blood cell line. Expected band seen at 65kD. Primary cGAS Monoclonal Antibody was used at 2µg/ml, blocking with 5% Milk in TBS-T. Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP (Product # 31430) was used at 1:10,000 dilution. Visualization was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34577).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-cGAS Monoclonal Antibody (HGAYN), eBioscience™ (Product # 14-5158-82) and a 60 kDa band corresponding to Cyclic GMP-AMP synthase was observed across all cell lines tested. Whole cell extracts (40 µg lysate) of HeLa (Lane 1), PANC-1 (Lane 2), BeWo (Lane 3), IMR-32 (Lane 4), SH-SY5Y (Lane 5) 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 µg/mL concentration) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 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)
Main image
Experimental details: Fig. 4 G3BP1 loss inhibits cGAS signaling required for SASP activation in senescent cells. WI-38 cells were treated with siRNA against G3BP1 (siG3BP1 #1 and #2) or scrambled control (siCTL) and assessed during proliferative stage (PRO) and 8-day post-ionizing radiation (+IR) a RNA was extracted and assayed by RT-qPCR using primers against IFNB1 mRNA. The data are representative of three independent experiments (two-tailed unpaired Student's t test). b (left) Lysates obtained from WI-38 cells +IR cell were subjected to western blot analysis against indicated proteins. (right) Quantifications represent a mean of relative protein levels from three independent experiments +- s.e.m (two-tailed unpaired Student's t test). c Immunofluorescence analysis against gammaH2AX and cGAS. DAPI staining was used to visualize nuclei. White arrows indicate CCF. Scale bar, 20 mum. d Graphs represent intensity profile for CCFs in (left) +IR siCTL and (right) +IR siG3BP1 treated WI-38 cells after immunofluorescence against gammaH2AX, cGAS, and DAPI. Plot is representative of foci shown in ( c ). Scale bar, 2 mum. e - g Graph of % SEN cells positive for CCF ( e ), number of CCFs present in SEN cells +- s.e.m ( f ), and number of CCFs present in SEN cells colocalized with cGAS ( g ). The data are representative of three independent experiments (two-tailed unpaired S tudent's t test, exact

14-5158-82