Western Blot Analysis of GIMAP Protein Family

Cells were lysed in radioimmunoprecipitation assay buffer (150 mM NaCl, 5 mM EDTA [pH 8.0], 50 mM Tris [pH 8.0], 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) complete with protease and phosphatase inhibitor cocktails, heated at 95°C for 5 min with reducing reagent, and run on a Bis-tris protein gel. Primary antibodies used were anti-β actin (clone 2D1D10; GenScript), anti-HA tag (clone 6E2; Cell Signaling Technologies), anti-LC3 rabbit polyclonal (cat no. L8919; Sigma-Aldrich), anti-human GIMAP1 (PA5-60858; Thermo Fischer Scientific), anti-human GIMAP4 (HPA019137; Sigma-Aldrich), anti-human GIMAP7 (HPA020268; Sigma-Aldrich), and anti-β tubulin Dylight 680 (clone BT7R; Life Technologies). Anti-human and anti-mouse GIMAP6 antibodies (MAC445 and MAC436, respectively), anti-human GIMAP2 antibody, and anti-human GIMAP8 antibody were sourced as previously described (Pascall et al., 2013 (link)). Secondary antibodies used were IRDye 800CW Donkey anti-Mouse (Murine) IgG, IRDye 800CW Donkey anti-Rabbit IgG, and IRDye 800CW Goat anti-Rat IgG (all from Li-COR Biosciences). Membranes were imaged using the Odyssey CLx Imaging System (LI-COR Biosciences) or PXi imager (Syngene). Data were analyzed using Image Studio Lite.

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Yao Y., Du Jiang P., Chao B.N., Cagdas D., Kubo S., Balasubramaniyam A., Zhang Y., Shadur B., NaserEddin A., Folio L.R., Schwarz B., Bohrnsen E., Zheng L., Lynberg M., Gottlieb S., Leney-Greene M.A., Park A.Y., Tezcan I., Akdogan A., Gocmen R., Onder S., Rosenberg A., Soilleux E.J., Johnson E., Jackson P.K., Demeter J., Chauvin S.D., Paul F., Selbach M., Bulut H., Clatworthy M.R., Tuong Z.K., Zhang H., Stewart B.J., Bosio C.M., Stepensky P., Clare S., Ganesan S., Pascall J.C., Daumke O., Butcher G.W., McMichael A.J., Simon A.K, & Lenardo M.J. (2022). GIMAP6 regulates autophagy, immune competence, and inflammation in mice and humans. The Journal of Experimental Medicine, 219(6), e20201405.

Publication 2022

IRDye 800CW Donkey anti-Rabbit IgG IRDye 800CW Goat anti-Rat IgG Odyssey CLx Imaging System PXi imager

Actin Anti antibodies Anti antibody Anti igg Antibodies Bis tris Buffer Cells Clone Donkey Edta Goat Human Irdye 800cw Membranes Murine Nacl Np 40 Phosphatase Protease Protein Rabbit Radioimmunoprecipitation assay Sodium deoxycholate Tris Tubulin

Corresponding Organization : Hacettepe University Hospital

Other organizations : University of Oxford, Max Delbrück Center, Freie Universität Berlin, Hadassah Medical Center, Garvan Institute of Medical Research, St Vincent's Clinic, UNSW Sydney, National Institutes of Health Clinical Center, National Institute of Diabetes and Digestive and Kidney Diseases, Johns Hopkins University, University of Cambridge, Stanford University, Baxter (United States), Charité - Universitätsmedizin Berlin, MRC Laboratory of Molecular Biology, Medical Research Council, Wellcome Sanger Institute, Babraham Institute

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Variable analysis

independent variables

Lysis buffer (radioimmunoprecipitation assay buffer) composition (150 mM NaCl, 5 mM EDTA [pH 8.0], 50 mM Tris [pH 8.0], 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS)
Heating at 95°C for 5 min with reducing reagent
Protein gel used (Bis-tris)

dependent variables

Protein expression levels of β actin, HA tag, LC3, GIMAP1, GIMAP4, GIMAP7, and β tubulin

control variables

Protease and phosphatase inhibitor cocktails added to lysis buffer
Primary and secondary antibodies used for detection

positive controls

Anti-β actin (clone 2D1D10; GenScript)
Anti-β tubulin Dylight 680 (clone BT7R; Life Technologies)

negative controls

Not explicitly mentioned

Annotations

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