- PA1-9073 - Invitrogen Antibodies NCF1 antibody

Musicki B, Karakus S, La Favor JD, Chen H, Silva FH, Sturny M, Zirkin BR, Burnett AL
Journal of cellular physiology 2021 Apr;236(4):3073-3082

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot detection of NCF1 in U937 lysate using Product # PA1-9073.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-p47phox Polyclonal Antibody (Product # PA1-9073) and a 50 kDa band corresponding to p47phox was observed across cell lines and tissue extract tested except A-431, PC-3, Mouse Adipose and Mouse Brain which are reported to be low. Membrane enriched extracts (30 µg lysate) of Ramos (Lane 1), Raji (Lane 2), Daudi (Lane 3), A-431 (Lane 4) and PC-3 (Lane 5) and tissue extracts of Mouse adipose (Lane 6), Mouse Brain (Lane 7) and Mouse Spleen (Lane 8) were electrophoresed using Novex® 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 (0.5 µg/mL) and detected by chemiluminescence with Rabbit anti-Goat IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27014, 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)
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Experimental details: Immunocytochemical analysis of p47phox in HeLa cells using a p47phox polyclonal antibody (Product #PA1-9073). HeLa cells were permeabilized with 0.15% Triton. Cells were incubated with 10 µg/mL of primary antibody for one hour followed by an Alexa Fluor 488 secondary antibody at a concentration of 2 µg/mL. Cytoplasmic staining can be seen as shown above. The nuclear stain is DAPI (blue). Negative control: Unimmunized goat IgG (10 µg/mL) followed by an Alexa Fluor 488 secondary antibody (2 µg/mL).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunocytochemical analysis of p47phox in HeLa cells using a p47phox polyclonal antibody (Product #PA1-9073). HeLa cells were permeabilized with 0.15% Triton. Cells were incubated with 10 µg/mL of primary antibody for one hour followed by an Alexa Fluor 488 secondary antibody at a concentration of 2 µg/mL. Cytoplasmic staining can be seen as shown above. The nuclear stain is DAPI (blue). Negative control: Unimmunized goat IgG (10 µg/mL) followed by an Alexa Fluor 488 secondary antibody (2 µg/mL).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of p47phox was performed using 70% confluent log phase Raji cells. 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 p47phox Goat Polyclonal Antibody (Product # PA1-9073) at 5 µg/mL in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Rabbit anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Product # A-11078) 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). Panel d represents the merged image showing cytoplasmic and membrane localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of p47phox in paraffin embedded human colon using a p47phox polyclonal antibody (Product #PA1-9073) at a concentration of 5 µg/mL. Steamed antigen retrieval was performed with pH 6 buffered citrate. Samples were then stained with alkaline phosphatase.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometric analysis of p47phox in HeLa cells using a polyclonal antibody (Product #PA1-9073). HeLa cells (blue line) were paraformaldehyde fixed and permeabilized with 0.5% Triton. The primary antibody was incubated for one hour (10 µg/mL) followed by an Alexa Fluor 488 secondary antibody (1 µg/mL). IgG control: Unimmunized goat IgG (black line) followed by an Alexa Fluor 488 secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 6 Figure Protein expressions of nitrotyrosine and p47phox subunit of NADPH oxidase were increased in the SCD compared to WT mouse penis and were decreased in the SCD mouse penis by FGIN-1-27 treatment. Representative western blots and quantification of nitrotyrosine (a) and p47phox (b) in penes isolated from WT and SCD mice treated with a TSPO ligand FGIN-1-27 or vehicle for nine days. Protein levels of nitrotyrosine and p47phox were analyzed by immunoblotting and normalized to GAPDH expression. Statistical analysis was performed by using one-way analysis of variance, followed by Newman-Keuls multiple comparison tests (between treatment groups) and by modified t test (between control and each treatment group). Data represent mean +- SEM ( n = 5-6). * p < .05 versus WT; # p < .05 versus SCD. FGIN-1-27, N , N -dihexyl-2-(4-fluorophenyl)indole-3-acetamide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SCD, sickle cell disease; TSPO, translocator protein; WT, wild-type

PA1-9073