- PA5-22101 - Invitrogen Antibodies MX1 antibody

Submitted references SARS-CoV-2 triggers an MDA-5-dependent interferon response which is unable to control replication in lung epithelial cells.

Rebendenne A, Valadão ALC, Tauziet M, Maarifi G, Bonaventure B, McKellar J, Planès R, Nisole S, Arnaud-Arnould M, Moncorgé O, Goujon C
Journal of virology 2021 Mar 25;95(8)

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of MX1 using 30 µg NCI-H929 whole cell lysate. Samples were loaded onto a 7.5% SDS-PAGE gel and probed with a MX1 polyclonal antibody (Product # PA5-22101) at a dilution of 1:1000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot analysis of MX1 was performed by separating 30 µg of various whole cell extracts by 7.5% SDS-PAGE. Proteins were transferred to a membrane and probed with a MX1 Polyclonal Antibody (Product # PA5-22101) at a dilution of 1:1000 and a HRP-conjugated anti-rabbit IgG secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot analysis of MX1 was performed by separating 30 µg of various whole cell extracts by 7.5% SDS-PAGE. Proteins were transferred to a membrane and probed with a MX1 Polyclonal Antibody (Product # PA5-22101) at a dilution of 1:1000 and a HRP-conjugated anti-rabbit IgG secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot using MX1 Polyclonal Antibody (Product # PA5-22101). Whole cell extract (30 µg) was separated by 7.5% SDS-PAGE, and the membrane was blotted with MX1 Polyclonal Antibody (Product # PA5-22101) diluted at 1:1,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: MX1 Polyclonal Antibody detects MX1 protein by western blot analysis. A. 30 µg 293T whole cell lysate/extract. B. 30 µg whole cell lysate/extract of human MX1-transfected 293T cells.7.5% SDS-PAGE. MX1 Polyclonal Antibody (Product # PA5-22101) dilution: 1:10,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using MX1 Polyclonal Antibody (Product # PA5-22101) and a 91 kDa band corresponding to MX1 was observed in cell lines treated with IFN alpha. Whole cell extracts (30 µg lysate) of Daudi (Lane 1), Daudi treated with IFN Alpha (10ng/mL, 16 hrs) (Lane 2), HeLa (Lane 3) and HeLa treated with IFN Alpha (10ng/mL, 16 hrs) were electrophoresed using NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX). 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 (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemistry-Immunofluorescence analysis of MX1 was performed in HeLa cells fixed in 4% paraformaldehyde at RT for 15 min. Green: MX1 Polyclonal Antibody (Product # PA5-22101) diluted at 1:200. Red: alpha Tubulin, a cytoskeleton marker. Blue: Hoechst 33342 staining.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of MX1 was performed using 70% confluent log phase HeLa and HeLa cells treated with IFN Alpha (10 ng/mL, 16hrs). The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with MX1 Polyclonal Antibody (Product # PA5-22101) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e represents untreated HeLa cells having no expression of MX1. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry (Paraffin) analysis of MX1 was performed in paraffin-embedded human colon carcinoma tissue using MX1 Polyclonal Antibody (Product # PA5-22101) at a dilution of 1:500.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemical analysis of paraffin-embedded MDA-MB-468 xenograft, using MX1 (Product # PA5-22101) antibody at 1:500 dilution. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: FIG 7 Inhibition of SARS-CoV-2 replication by type I IFN pretreatment in primary HAE cells and immortalized Calu-3 cells. (A) Human HAE cells (nasal, tracheal, or bronchial, as indicated) were pretreated or not with type I IFN for 20 h, and N.I. or incubated with SARS-CoV-2 on the apical side at MOI 0.1 for 1 to 2 h. Cells were harvested at 72 h postinfection, then lysed for RNA extraction and RdRp RT-qPCR analysis. (B) Plaque assays were performed on washes of the apical side of the HAE cells from (A) at 24 h, 48 h, and 72 h to determine the number of PFU per ml of supernatant (gray dotted line = detection threshold). (C) Human HAE cells were pretreated or not with IFN for 20 h, and N.I. or incubated with SARS-CoV-2 on the apical side at MOI 0.1 and 0.25 for 2 h. After 48 h, cells were stained for actin with phalloidin (magenta) and an anti-double-stranded RNA antibody (green). Representative images, acquired with an LSM880 Airyscan microscope, are shown; scale bar 10 mum. D. Calu-3 cells were pretreated or not with IFN for 16 to 20 h, the cells were N.I. or incubated with SARS-CoV-2 at the indicated MOIs, and lysed 24 h postinfection for immunoblot analysis of SARS-CoV-2 nucleoprotein (N) and spike, and IFITM3, RIG-I, MX1, and actin expression levels. A representative immunoblot is shown. (E) Human Calu-3 cells were pretreated or not with IFN, and infected as in (D). Cells were harvested and lysed for RNA extraction and RdRp RT-qPCR analysis. (F) Production of infectious vi

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIG 8 Inhibition of SARS-CoV-2 replication by type I IFN in A549-ACE2 and Caco-2-ACE2 cells. Human A549-ACE2 (A, C, and E) and Caco-2-ACE2 (B, D, and F) cells, as indicated, were pretreated or not with IFN for 16 to 20 h, then the medium was replaced and cells were mock infected (N.I.) or incubated with SARS-CoV-2 at the indicated MOIs. (A and B) Cells were harvested and lysed for RNA extraction and RT-qPCR analysis using RdRp primers and probe. (C and D) Cells were fixed with PFA, permeabilized, and stained with an anti-spike antibody conjugated to an Alexa fluorochrome. The percentage of spike(+) cells was scored by flow cytometry. (E and F) Cells were lysed for immunoblot analysis of SARS-CoV-2 nucleoprotein (N) and spike, IFITM3, RIG-I, and MX1 ISG expression levels. Actin serves as a loading control. Representative immunoblots are shown. Of note, MX1 was not detected in Caco-2-ACE2 cell lysates. The means of three independent experiments are shown (A to D), with error bars representing one standard deviation (SD) from the mean.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: FIG 9 IFN production upon SARS-CoV-2 replication does not protect Calu-3 and A549-ACE2 cells against infection. (A) CTRL, RIG-I , MDA-5 , and MAVS Calu-3 knockout (KO) cells were infected with SARS-CoV-2 at MOI 0.05 (as in Fig. 5B and C ) and viral production was measured 48 h later by plaque assays on Vero E6 cells. (B) CTRL and IRF9 Calu-3 KO cells were generated and selected. Cells were infected with SARS-CoV-2 at the indicated MOIs and viral replication was evaluated 48 h later by RdRp RT-qPCR. (C) CTRL and IRF9 KO cells were pretreated or not with IFN for 48 h, lysed, and the expression levels of IFITM3, RIG-I, MX1, and actin were analyzed by immunoblotting. (D) CTRL and JAK1 A549-ACE2 knockout cells were generated and selected. Cells were infected with SARS-CoV-2 at MOI 0.0005 and viral replication was measured 48 h later using RdRp RT-qPCR. (E) CTRL and JAK1 A549-ACE2 knockout cells were pretreated or not with IFN for 48 h, lysed, and the expression levels of IFITM3, RIG-I, and MX1 were analyzed by immunoblotting, with actin serving as a loading control. (F) Calu-3 cells were infected with SARS-CoV-2 at the indicated MOIs after a 24-h pretreatment with IFN or not, or were subsequently treated with IFN at 4 h, 8 h, or 24 h postinfection. Viral replication was measured at 48 h postinfection using RdRp RT-qPCR. The means of two (A and D) or three (B and F) independent experiments are shown, with error bars representing the SD. Representative immunoblots (C and E) are show

PA5-22101

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