- PA5-36773 - Invitrogen Antibodies NCF1 antibody

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

EphA2 Is a Neutrophil Receptor for Candida albicans that Stimulates Antifungal Activity during Oropharyngeal Infection.

Swidergall M, Solis NV, Wang Z, Phan QT, Marshall ME, Lionakis MS, Pearlman E, Filler SG
Cell reports 2019 Jul 9;28(2):423-433.e5

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of Phospho-p47phox pSer304 in paraffin-embedded human colorectal carcinoma using Phospho-p47phox pSer304 polyclonal antibody (Product # PA5-36773) at a dilution of 1:50.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of Phospho-p47phox (Ser304) in paraffin-embedded human colorectal carcinoma tissue. Samples were incubated with Phospho-p47phox (Ser304) polyclonal antibody (Product # PA5-36773) at a dilution of 1:50.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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)

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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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

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5. EphA2 Activates the MEK-ERK MAPK Module to Prime p47 phox (A) Intracellular ROS accumulation measured by mean fluorescence (FL) intensity in wild-type and EphA2 -/- neutrophils after45 min of infection with opsonized C. albicans yeast. Results are median +- interquartile range of neutrophils from 3 mice per strain, each tested in duplicate. ****p < 0.0001 (Mann-Whitney test corrected for multiple comparisons). (B) Effects of EphA2 on localization of p47 phox in neutrophils infected with C. albicans . Wild-type and EphA2 -/- neutrophils were incubated with opsonized GFP-expressing C. albicans yeast for 30 min, fixed, and stained for p47 phox (red) and F-actin (blue). Scale bar, 10 mum. (C) Percentage of phagosomes containing C. albicans and surrounded by p47 phox . Results are median +- interquartile of 60 neutrophils per mouse strain in three independent experiments. ****p < 0.01 (Mann-Whitney test). (D) Representative immunoblot of MEK 1/2 and ERK 1/2 phosphorylation in wild-type and EphA2 -/- neutrophils that had been infected with yeast-phase C. albicans for 30 min. (E) Representative immunoblot of PKC-delta phosphorylation in wild-type and EphA2 -/- neutrophils that had been infected with yeast-phase C. albicans SC5314 for 30 min with a 5:1 ratio. (F) Representative immunoblot of ERK 1/2 phosphorylation in wild-type and EphA2 -/- neutrophils stimulated with 50 nM PMA for 30 min. (G) Intracellular ROS accumulation (mean fluorescence [FL] intensity) of wild-type a

PA5-36773

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