Irdye 680lt donkey anti mouse igg h l

Manufactured by LI COR

IRDye 680LT donkey anti-mouse IgG (H + L) is a fluorescent secondary antibody used for detection of mouse primary antibodies in Western blotting, ELISA, and other immunoassays. The antibody is conjugated with the IRDye 680LT infrared dye, which provides a stable and bright signal.

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3 protocols using irdye 680lt donkey anti mouse igg h l

Western Blot for Apoptosis Markers

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The proteins were extracted from cells using RIPA lysis buffer (89900, Thermo Scientific™) containing Protease Inhibitor Cocktail (78441, Thermo Scientific™). Proteins were separated by 8%–13% SDS-PAGE gel and subsequently transferred to polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with blocking buffer (37535, Thermo Scientific™) for 1 h at room temperature. After blocking, membranes were incubated over night at 4 °C with diluted primary antibodies against caspase-3 (14220S, Cell Signaling Technology), cleaved caspase-3 (9664S, Cell Signaling Technology), PARP (9532S, Cell Technology), GAPDH (10R-G109a, Fitzgerald Industries), and Actin (A5441, Sigma-Aldrich). The secondary antibodies used were IRDye 800CW donkey anti-rabbit IgG (H + L) and IRDye 680LT donkey anti-mouse IgG (H + L) (LI-COR Biosciences). The membranes were scanned on an Odyssey imaging system (LI-COR Biosciences).

You W., Zhou T., Knoops K., Berendschot T.T., van Zandvoort M.A., Germeraad W.T., Benedikter B., Webers C.A., Reutelingsperger C.P, & Gorgels T.G. (2023). Stressed neuronal cells can recover from profound membrane blebbing, nuclear condensation and mitochondrial fragmentation, but not from cytochrome c release. Scientific Reports, 13, 11045.

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Western Blot Analysis of Signaling Proteins

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Cells were lysed in Laemmli sample buffer and run in 4–20% Mini-PROTEAN TGX precast protein gels (Bio-Rad Laboratories). The primary antibodies used were rabbit anti-Egr1 (no. 4154; Cell Signaling), rabbit anti–c-Fos (no. 2250; Cell Signaling), rabbit anti–c-Jun (no. 9165; Cell Signaling), mouse anti-Snail (no. 3895; Cell Signaling), rabbit anti-PARP1 (no. 9532; Cell Signaling), rabbit anti-Smad2/3 (no. 8685; Cell Signaling), rabbit anti-pSmad2/3 (no. 8828; Cell Signaling), mouse anti–cytochrome c (no. sc-13560; Santa Cruz Biotechnology), mouse anti-Cox4 (no. 11967; Cell Signaling), rabbit anti–caspase 9 (no. 9502; Cell Signaling), mouse anti–caspase 8 (no. 9746; Cell Signaling), rabbit anti-Tubulin (no. 2128; Cell Signaling), and mouse anti–α-Tubulin (T6199; Sigma). The secondary antibodies used were IRDye 800CW donkey anti–rabbit IgG (H+L), IRDye 680LT donkey anti–mouse IgG (H+L), and IRDye 800CW donkey anti–mouse IgG (H+L; LI-COR Biosciences). The blots were scanned on an Odyssey imaging system (LI-COR Biosciences). Cell treatment, sample collection, and Western blotting were repeated at least three times, and the representative blots are shown in the figures.

Sun G., Guzman E., Balasanyan V., Conner C.M., Wong K., Zhou H.R., Kosik K.S, & Montell D.J. (2017). A molecular signature for anastasis, recovery from the brink of apoptotic cell death. The Journal of Cell Biology, 216(10), 3355-3368.

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Immunoblotting of NRG1 and Downstream Signaling

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Immunoblotting was performed as described previously (1 (link)). The following antibodies were used: anti-NRG1 β1 (R&D, AF-396-NA), anti-CD74 (Abcam, ab22604), anti-HSP90 (Cell Signaling Technology, No. 4877), anti-ERBB3 (Cell Signaling Technology, No. 4754), anti-phosphoERBB3 (Cell Signaling Technology, No. 4791), ERK1/2 (Cell Signaling Technology, No. 9102), anti-phosphoERK1/2 (Cell Signaling Technology, No. 9106), anti-AKT (Cell Signaling Technology, No. 9272), anti-phosphoAKT (Cell Signaling Technology, No. 9271), and β-actin-HRP (Santa Cruz Biotechnology, sc-47778). Secondary antibodies were IRDye800CW donkey anti-goat IgG (H+L; Licor, 925–32214), IRDye 680LT donkey anti-mouse IgG (H+L; Licor, 925–68022), and IRDye 800CW goat anti-rabbit IgG (H+L; Licor, 926–32211). Fluorescence detection was performed on Odyssey CLx Imaging System (Licor).

Werr L., Plenker D., Dammert M.A., Lorenz C., Brägelmann J., Tumbrink H.L., Klein S., Schmitt A., Büttner R., Persigehl T., Shokat K.M., Wunderlich F.T., Schram A.M., Peifer M., Sos M.L., Reinhardt H.C, & Thomas R.K. (2022). CD74-NRG1 Fusions Are Oncogenic In Vivo and Induce Therapeutically Tractable ERBB2:ERBB3 Heterodimerization. Molecular Cancer Therapeutics, 21(5), 821-830.

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