Antibody data
- Antibody Data
- Antigen structure
- References [1]
- Comments [0]
- Validations
- Western blot [3]
- Other assay [4]
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- Product number
- PA5-31169 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- p47phox Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Recombinant protein fragment
- Description
- Recommended positive controls: Raji, mouse spleen. Predicted reactivity: Mouse (84%), Rat (82%), Pig (84%), Rabbit (86%), Bovine (88%). Store product as a concentrated solution. Centrifuge briefly prior to opening the vial.
- Reactivity
- Human, Mouse
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µL
- Concentration
- 1 mg/mL
- Storage
- Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.
Submitted references Shear and Integrin Outside-In Signaling Activate NADPH-Oxidase 2 to Promote Platelet Activation.
Xu Z, Liang Y, Delaney MK, Zhang Y, Kim K, Li J, Bai Y, Cho J, Ushio-Fukai M, Cheng N, Du X
Arteriosclerosis, thrombosis, and vascular biology 2021 May 5;41(5):1638-1653
Arteriosclerosis, thrombosis, and vascular biology 2021 May 5;41(5):1638-1653
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western Blot using p47phox Polyclonal Antibody (Product # PA5-31169). Sample (30 µg of whole cell lysate). Lane A: Raji. 10% SDS PAGE. p47phox Polyclonal Antibody (Product # PA5-31169) diluted at 1:5,000.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western Blot using p47phox Polyclonal Antibody (Product # PA5-31169). Sample (50 µg of whole cell lysate). Lane A: mouse spleen. 10% SDS PAGE. p47phox Polyclonal Antibody (Product # PA5-31169) diluted at 1:500.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Anti-p47phox Polyclonal Antibody (Product # PA5-31169) and a 50 kDa band corresponding to p47phox was observed in Raji and Daudi but was absent in A-431 and PC-3 which are reported to be low. A non-specific band around 70 kDa was also observed across cell lines. Membrane enriched extracts (30 µg lysate) of Raji (Lane 1), Daudi (Lane 2), A-431 (Lane 3) and PC-3 (Lane 4) 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 (1:3000 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
- Figure 2. Effect of integrilin and importance of integrin alpha IIb beta 3 in activation of p47phox during platelet activation. A and B , Washed wild-type (WT) or beta 3 -/- mouse platelets, or WT mouse platelets treated with vehicle control (citric acid [CA]) or integrilin (10 ug/mL), were stimulated with or without thrombin (0.025 U/mL; A ), CRP (collagen-related peptide; ug/mL; B ) under shear, and were lysed and analyzed via SDS-PAGE and Western Blot for phosphorylated p47phox at Ser 304 (pS304-p47phox), total p47phox, or alpha-tubulin as loading control. C and D , Lysates of washed WT and beta 3 -/- mouse platelets stimulated with increasing doses of thrombin ( C ) or CRP ( D ) were probed for phosphorylated p47phox at Ser 304 (pS304-p47phox), total p47phox or alpha-tubulin as loading control. Lower parts of A-D are quantifications of Western blot results. Data plotted as mean+-SEM (n=3, * P
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Figure 3. Reactive oxygen species (ROS) production and p47phox activation during platelet spreading on integrin ligand fibrinogen. A , Images of DCF fluorescence in mouse platelets loaded with 10 umol/L carboxy-H 2 DCFDA and allowed to spread on fibrinogen, with or without 0.01 U/mL thrombin. N-acetylcysteine (NAC)-treated platelets was used as negative control (scale bar, 10 um). B , Quantification of the total ROS production per cell was calculated by integrating the fluorescence (FL) intensity (mean+-SEM, * P
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Figure 4. Galpha13-mediated integrin outside-in signaling stimulates reactive oxygen species (ROS) production, p47phox activation and Syk phosphorylation. A , Images of DCF fluorescence in mouse platelets loaded with 10 umol/L carboxy-H 2 DCFDA, treated with 100 umol/L scrambled control peptide or mP6, and allowed to spread on fibrinogen with or without 0.01 U/mL thrombin (scale bar, 10 um). B , Quantification of the total ROS production per cell in ( A ) was calculated by integrating the fluorescence (FL) intensity (mean+-SEM, Kruskal-Wallis test with Dunn multiple comparisons, Fg -thrombin, Scr: n=100, Fg -thrombin, mP6: n=6; Fg+thrombin, Scr: n=45, Fg+thrombin, mP6 n=9). C and D , Washed mouse platelets were treated with 100 umol/L scrambled control peptide or mP6, allowed to adhere and spread on fibrinogen, and lysed for ( C ) SDS-PAGE and Western blot for phosphorylated p47phox at Ser 304 (pS304-p47phox), total p47phox or alpha-tubulin as loading control. D , Quantification of ( C ), plotted as mean+-SEM, Student t test, n=4). E and F , Washed mouse platelets, treated with 100 umol/L scrambled control peptide or mP6 ( E ), or wild type and Galpha 13 -/- platelets ( F ), were preincubated with 10 umol/L carboxy-H 2 DCFDA and stimulated with 0.025 U/mL thrombin under the shear rate of 800/s, and total ROS production is shown as mean+-SEM (Student t test, n=3). G and H , Mouse platelets treated with scrambled control peptide or mP6 were stimulated with thrombin (0.025U/mL)
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Figure 6. Differential roles of SFKs (Src family kinases) and Syk in agonist-induced p47phox phosphorylation. A and B , Washed mouse platelets were treated with the SFK inhibitor PP2 (10 umol/L) or negative control compound PP3(10 umol/L), and stimulated with ( A ) 0.025 U/mL thrombin or ( B ) 1 ug/mL CRP (collagen-related peptide) to induce aggregation. C and D , Washed mouse platelets were treated with DMSO control (ctrl) or the Syk inhibitor piceatannol (15 umol/L) and stimulated with ( C ) 0.025 U/mL thrombin or ( D ) 1 ug/mL CRP to induce aggregation. After 2 min, platelets were lysed and analyzed via SDS-PAGE and Western Blot for phosphorylated p47phox at Ser 304 (p-p47phox S 304 ) or Ser 328 (p-p47phox S 328 ), total p47phox or alpha-tubulin as loading control. The blots were scanned and quantified using National Institutes of Health Image J and plotted as mean+-SEM (* P