- 700360 - Invitrogen Antibodies IFIH1 antibody

Elsaid AF, Agrawal S, Agrawal A, Ghoneum M
Nutrients 2021 Nov 19;13(11)

Zhou X, Backman LJ, Danielson P
Scientific reports 2021 Apr 1;11(1):7360

Shao W, Earley LF, Chai Z, Chen X, Sun J, He T, Deng M, Hirsch ML, Ting J, Samulski RJ, Li C
JCI insight 2018 Jun 21;3(12)

Nikolic J, Civas A, Lama Z, Lagaudrière-Gesbert C, Blondel D
PLoS pathogens 2016 Oct;12(10):e1005942

Israelow B, Narbus CM, Sourisseau M, Evans MJ
Hepatology (Baltimore, Md.) 2014 Oct;60(4):1170-9

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of MDA5 in MDA5-transfected 293 cells (right) and untransfected cells (left) using a MDA5 recombinant rabbit monoclonal antibody (Product # 700360) at a dilution of 5 µg/mL followed by detection using an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody at a dilution of 1:1000 (green) and nuclei stained using Hoescht (blue).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of MDA5 in MDA5-transfected 293 cells (right) and untransfected cells (left) using a MDA5 recombinant rabbit monoclonal antibody (Product # 700360) at a dilution of 5 µg/mL followed by detection using an Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody at a dilution of 1:1000 (green) and nuclei stained using Hoescht (blue).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of MDA5 was done on 70% confluent log phase A431 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with MDA5 (33H12L34), Recombinant Rabbit Monoclonal Antibody (Product # 700360) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing cytoplasmic and nuclear localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of MDA5 was done on MCF7 cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with MDA5 Rabbit Monoclonal Antibody (700360, red histogram) or with rabbit isotype control (yellow histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: RIG-I like receptor and IFN-lambda receptor silencing promotes HCV multicycle growth in HepG2-HFL cells. (A) Immunoblots of endogenous RIG-I, MDA5, or ß-actin were performed on lysates from HepG2-HFL cells that were transduced with lentiviral vectors expressing shRNA targeting RIG-I, MDA5, or a no targeting (NT) shRNA used as a negative control. (B) IL-29 production was assayed by ELISA 24 hpi with SeV and 24 hours transfectionwith poly(I:C). (C) HCV-GLuc multicycle growth was monitored by sampling luciferase production at 2, 4, and 7 dpi. (D) IL-29 mRNA induction was assessed in HepG2-HFL cells 30 hpi with non-reporter HCV. (E) Quantification of IFN-lambda receptor (IL28RA) mRNA levels in HepG2-HFL cells transduced with two different lentiviral vectors expressing shRNA targeting IL28RA (#1 and #2), or a control shRNA (NT). (F) HCV-GLuc multicycle growth was monitored by sampling luciferase production at 2, 4, and 7 dpi. Asterisks represent statistically significant differences between NT-shRNA expressing cells and cells expressing shRNA targeting RIG-I, MDA5, or IL-28RA at the indicated time points.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of MDA5 was performed in K562 cells. Antigen-antibody complexes were formed by incubating approximately 500 µg whole cell lysate with 5 to 10 µL of recombinant monoclonal MDA5 antibody (Product # 700360) rotating 60 min at RT. The immune complexes were captured on 625 µg of anti- rabbit coated Dynabeads (Product # 11204D) and washed extensively. They were then eluted and analyzed using the Simple Western system using the same antibody as used in immunoprecipitation at a dilution of 1:25, followed by a 1:100 dilution of secondary antibody. Lane 1 is the input, lane 2 no antibody IP and lane 3 is the target specific IP. Data courtesy of the Yeo lab as part of the ENCODE project.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 MDA5 signaling induces IL-8 upregulation via NF-kappaB phosphorylation in CD+ keratocytes. ( A ) Western blot of NF-kappaB phosphorylation in CD- and CD+ keratocytes with/without mitoTEMPO ( n = 4). NF-kappaB and p-NF-kappaB were blotted and cropped from the same gel. p-NF-kappaB was blotted first and the membrane was stripped and washed for NF-kappaB blotting. Actin was blotted separately from a different gel using the corresponding samples. Exposures were adjusted automatically by the Odyssey Fc imaging system. ( B ) DDX58 and IFIH1 mRNA expression in CD+/- keratocytes 48 h after respective siRNA transfection ( n = 3). ( C ) IL-8 mRNA expression in keratocytes 24 h after 20 nM TPCA treatment ( n = 3). ( D ) NF-kappaB phosphorylation, analyzed by western blot, after IFIH1 knock-down in CD+/- keratocytes ( n = 3). NF-kappaB and p-NF-kappaB were blotted and cropped from the same gel. p-NF-kappaB was blotted first and the membrane was stripped and washed for NF-kappaB blotting. GAPDH and MDA5 were blotted separately from a different gel using the corresponding samples. Exposures were adjusted automatically by the Odyssey Fc imaging system. ( E ) IL-8 mRNA expression in CD+/- keratocytes after IFIH1 knockdown by siRNA transfection ( n = 3). Values are means +- SD. n.s. (not significant); *P < 0.05, **P < 0.01.

700360