Mda 5 d74e4

Manufactured by Cell Signaling Technology

MDA-5 (D74E4) is a rabbit monoclonal antibody that recognizes the Melanoma Differentiation-Associated protein 5 (MDA-5). MDA-5 is a cytoplasmic receptor that plays a key role in the detection of viral double-stranded RNA, triggering an antiviral innate immune response.

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Lab products found in correlation

Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions) Isg15, by Cell Signaling Technology (1 mentions) Protease inhibitor mixture, by Merck Group (1 mentions) Bca protein concentration kit, by Beyotime (1 mentions) Ezh2 d2c9, by Cell Signaling Technology (1 mentions) Gapdh 14c10, by Cell Signaling Technology (1 mentions) Bca assay, by Thermo Fisher Scientific (1 mentions) Cgas d3o8o, by Cell Signaling Technology (1 mentions) Odyssey clx, by LI COR (1 mentions) Precellys 24 homogenizer, by Bertin Technologies (1 mentions) Polyclonal goat igg, by R&D Systems (1 mentions) Rig 1 d14g6, by Cell Signaling Technology (1 mentions) Rabbit polyclonal igg, by Thermo Fisher Scientific (1 mentions) Ripk1 38 rip, by BD (1 mentions) Gsk 843, by GlaxoSmithKline (1 mentions) Mlkl mabc604, by Merck Group (1 mentions) Cleaved caspase 3 clone d3e9, by Cell Signaling Technology (1 mentions) Anti fkbp12, by Thermo Fisher Scientific (1 mentions) Caspase 3 9662, by Cell Signaling Technology (1 mentions) Anti flag m2, by Merck Group (1 mentions)

4 protocols using mda 5 d74e4

Western Blot Analysis of Immune Proteins

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Cells were collected in RIPA lysis buffer (Thermo Scientific, cat#89900) containing protease inhibitor mixture (Sigma, cat#P-8340). Protein concentration was quantified using a BCA Protein concentration Kit (Beyotime, cat#P0009). Total cell lysates were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride membrane. The membrane was incubated with primary antibodies (overnight at 4°C) and sequentially horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 hour. Membranes were probed with HRP-conjugated secondary mouse antibody (Santa Cruz, cat#sc-516102) at room temperature for 1h. Enhanced chemiluminescence substrates (ECL) (Tanon, cat#180501) were used to visualize the protein abundances. Primary antibodies used were EZH2 (D2C9) (Cell Signaling Technology, cat#5246), TLR3 (TLR3.7) (Santacruz, cat#sc-32232), MDA-5 (D74E4) (Cell Signaling Technology, cat#5321), ISG15 (Cell Signaling Technology, cat#2743), Actin (Santacruz, cat#sc-8432).

Qiu F., Yang Q., Sun W., Ruan K., Jiang N, & Zhou J. (2022). EZH2 inhibition activates dsRNA-interferon axis stress and promotes response to PD-1 checkpoint blockade in NSCLC. Journal of Cancer, 13(9), 2893-2904.

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Snap-frozen Tumor Protein Analysis

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Snap frozen tumour samples were lysed in RIPA buffer using a Precellys 24 homogenizer (Bertin Technologies). All lysates were quantified by BCA assay (Thermo Fisher) prior to SDS-PAGE and western blotting. The following antibodies were used: GAPDH 14C10, MDA5 D74E4 and cGAS D3O8O (Cell Signaling). Secondary was IRDye 800CW Goat anti-Rabbit IgG H + L and imaged using a LI-COR Odyssey CLx (LI-COR Biosciences).

Dillon M.T., Bergerhoff K.F., Pedersen M., Whittock H., Crespo-Rodriguez E., Patin E E.C., Pearson A., Smith H.G., Paget J.T., Patel R.R., Foo S., Bozhanova G., Ragulan C., Fontana E., Desai K., Wilkins A.C., Sadanandam A., Melcher A., McLaughlin M, & Harrington K.J. (2019). ATR inhibition potentiates the radiation induced inflammatory tumor microenvironment. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(11), 3392-3403.

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Comprehensive Antibody Protocol for Cytometry and Western Blotting

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Flow cytometry antibodies are listed in Supplementary Table 1 and
immunohistochemistry antibodies are listed above. For western blotting, primary
antibodies against ADAR1 (15.8.6, Santa Cruz Biotechnology), PKR (EPR19374
Abcam), RIG-I (D14G6, Cell Signaling Technology), MDA5 (D74E4, Cell Signaling
Technology), STAT1 (p91, Polyclonal Goat IgG, R&D Systems), and MAVS (Rabbit
Polyclonal IgG, ThermoFisher Scientific)were used. Peroxidase-conjugated
secondary antibodies against rabbit IgG, mouse IgG or goat IgG were purchased
from Jackson Laboratories. IRDye secondary antibodies against rabbit IgG, mouse
IgG or goat IgG were purchased from LI-COR Biosciences.

Ishizuka J.J., Manguso R.T., Cheruiyot C.K., Bi K., Panda A., Vellve A.I., Miller B.C., Du P.P., Yates K.B., Dubrot J., Buchumenski I., Comstock D.E., Brown F.D., Ayer A., Kohnle I.C., Pope H.W., Zimmer M.D., Sen D.R., Lane-Reticker S.K., Robitschek E.J., Griffin G.K., Collins N.B., Long A.H., Doench J.G., Kozono D., Levanon E.Y, & Haining W.N. (2018). Loss of ADAR1 in tumours overcomes resistance to immune checkpoint blockade. Nature, 565(7737), 43-48.

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Multimodal Analysis of Cell Death Pathways

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Where indicated, the following drugs were used at the listed concentrations: 50 μM zVAD (SM Biochemicals), 100nM GSK’843 (GlaxoSmithKline).
The following antibodies were used for Western Blots and immunoprecipitations: ADAR1 (15.8.6) SantaCruz, ZBP1 (Zippy-1) AdipoGen, actin (13E5) Cell Signaling Technology, MDA5 (D74E4) Cell Signaling Technologies, p-RIPK3 (GEN135–35-9), Genentech, RIPK3 (1G6.1.4) Genentech, or RIPK3 (2283) ProSci, p-MLKL (D6E3G) Cell Signaling Technologies, MLKL (MABC604) Millipore, Caspase-3 (9662) Cell Signaling Technologies, Cleaved Capsase-3 (9661) Cell Signaling Technologies, RIPK1 (38/RIP) BD Biosciences, anti-FLAG (M2) Sigma, anti-FKBP12, Thermo Fisher (PA1–026A). Iba1 (Cat no. 019–19741) Wako-Chem, Cleaved Caspase 3 (Clone D3E9) Cell Signaling Technologies were used in immunohistochemical analysis.
The following antibodies were used for flow cytometry analysis of splenocytes: FITC anti-CD19 (clone 1D3; BD Biosciences) PerCP-Cy5.5 anti-CD3e (clone 145–2C11; BD Biosciences), PE-Cy7 anti-Ly6C (clone HK1.4; Biolegend), APC anti-F4/80 (clone BM8; eBioscience), AF700 anti-Ly6G (clone 1A8; Biolegend), APC-Cy7 anti-NK1.1 (clone PK136; BD Biosciences), BV510 anti-CD8a (clone 53–6.7; BD Biosciences); BV605 anti-CD4 (clone RM4–5; BD Biosciences), BV650 anti-CD11b (clone M1/70; Biolegend), and BUV395 anti-CD45.2 (clone 104; BD Biosciences).

Hubbard N.W., Ames J.M., Maurano M., Chu L.H., Somfleth K.Y., Gokhale N.S., Werner M., Snyder J.M., Lichauco K., Savan R., Stetson D.B, & Oberst A. (2022). ADAR1 mutation causes ZBP1-dependent immunopathology. Nature, 607(7920), 769-775.

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