Antibody data
- Antibody Data
- Antigen structure
- References [8]
- Comments [0]
- Validations
- Western blot [1]
- Immunocytochemistry [1]
- Other assay [6]
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- Product number
- PA5-67520 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Phospho-Nrf2 (Ser40) Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- Predicted to react with Mouse and Rat samples.
- Reactivity
- Human, Mouse, Rat
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 100 µL
- Concentration
- 1 mg/mL
- Storage
- -20°C
Submitted references Rumen-protected methionine during heat stress alters mTOR, insulin signaling, and 1-carbon metabolism protein abundance in liver, and whole-blood transsulfuration pathway genes in Holstein cows.
Antioxidant activity and laxative effects of tannin-enriched extract of Ecklonia cava in loperamide-induced constipation of SD rats.
Retinal oxidative stress activates the NRF2/ARE pathway: An early endogenous protective response to ocular hypertension.
α-Synuclein pathology in Parkinson disease activates homeostatic NRF2 anti-oxidant response.
The effects of meldonium on the acute ischemia/reperfusion liver injury in rats.
Deficiency of optineurin enhances osteoclast differentiation by attenuating the NRF2-mediated antioxidant response.
Methionine supply alters mammary gland antioxidant gene networks via phosphorylation of nuclear factor erythroid 2-like 2 (NFE2L2) protein in dairy cows during the periparturient period.
Phosphorylation of nuclear factor erythroid 2-like 2 (NFE2L2) in mammary tissue of Holstein cows during the periparturient period is associated with mRNA abundance of antioxidant gene networks.
Coleman DN, Totakul P, Onjai-Uea N, Aboragah A, Jiang Q, Vailati-Riboni M, Pate RT, Luchini D, Paengkoum P, Wanapat M, Cardoso FC, Loor JJ
Journal of dairy science 2022 Sep;105(9):7787-7804
Journal of dairy science 2022 Sep;105(9):7787-7804
Antioxidant activity and laxative effects of tannin-enriched extract of Ecklonia cava in loperamide-induced constipation of SD rats.
Kim JE, Choi YJ, Lee SJ, Gong JE, Lee YJ, Sung JE, Jung YS, Lee HS, Hong JT, Hwang DY
PloS one 2021;16(2):e0246363
PloS one 2021;16(2):e0246363
Retinal oxidative stress activates the NRF2/ARE pathway: An early endogenous protective response to ocular hypertension.
Naguib S, Backstrom JR, Gil M, Calkins DJ, Rex TS
Redox biology 2021 Jun;42:101883
Redox biology 2021 Jun;42:101883
α-Synuclein pathology in Parkinson disease activates homeostatic NRF2 anti-oxidant response.
Delaidelli A, Richner M, Jiang L, van der Laan A, Bergholdt Jul Christiansen I, Ferreira N, Nyengaard JR, Vægter CB, Jensen PH, Mackenzie IR, Sorensen PH, Jan A
Acta neuropathologica communications 2021 Jun 6;9(1):105
Acta neuropathologica communications 2021 Jun 6;9(1):105
The effects of meldonium on the acute ischemia/reperfusion liver injury in rats.
Đurašević S, Stojković M, Sopta J, Pavlović S, Borković-Mitić S, Ivanović A, Jasnić N, Tosti T, Đurović S, Đorđević J, Todorović Z
Scientific reports 2021 Jan 14;11(1):1305
Scientific reports 2021 Jan 14;11(1):1305
Deficiency of optineurin enhances osteoclast differentiation by attenuating the NRF2-mediated antioxidant response.
Xue P, Hu X, Chang E, Wang L, Chen M, Wu TH, Lee DJ, Foster BL, Tseng HC, Ko CC
Experimental & molecular medicine 2021 Apr;53(4):667-680
Experimental & molecular medicine 2021 Apr;53(4):667-680
Methionine supply alters mammary gland antioxidant gene networks via phosphorylation of nuclear factor erythroid 2-like 2 (NFE2L2) protein in dairy cows during the periparturient period.
Han L, Batistel F, Ma Y, Alharthi ASM, Parys C, Loor JJ
Journal of dairy science 2018 Sep;101(9):8505-8512
Journal of dairy science 2018 Sep;101(9):8505-8512
Phosphorylation of nuclear factor erythroid 2-like 2 (NFE2L2) in mammary tissue of Holstein cows during the periparturient period is associated with mRNA abundance of antioxidant gene networks.
Han LQ, Zhou Z, Ma Y, Batistel F, Osorio JS, Loor JJ
Journal of dairy science 2018 Jul;101(7):6511-6522
Journal of dairy science 2018 Jul;101(7):6511-6522
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Supportive validation
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- Invitrogen Antibodies (provider)
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- Experimental details
- Western blot analysis of Phospho-Nrf2 (Ser40) in HT-29 whole cell lysates using a Phospho-Nrf2 (Ser40) Polyclonal Antibody (Product # PA5-67520).
Supportive validation
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- Invitrogen Antibodies (provider)
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- Experimental details
- Immunofluorescence analysis of phospho-Nuclear factor erythroid 2-related factor 2 (Ser40) was performed using 70% confluent log phase Hep G2 treated with TBHQ (100 micromolar for 4h). 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 45 minutes at room temperature. The cells were labeled with Phospho-Nrf2 (Ser40) Polyclonal Antibody (Product # PA5-67520) at 1:100 dilution in 0.1% BSA, incubated at 4-degree Celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000 dilution), 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 dilution). Panel d represents the merged image showing nuclear localization. Panel e represents untreated cells showing low expression of phospho-NRF2 (ser40). Panel f represents control cells with no primary antibody to assess the background. The images were captured at 60X magnification.
Supportive validation
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- Fig 2 Antioxidant activity of TEE in vitro . (A) Determination of intracellular ROS production. After DCF-DA treatment into pRISMCs of each subset group, green fluorescence-stained cells were observed and counted using a fluorescent microscope, at 200x magnification. The fluorescence intensity was measured by ELISA reader. (B) Detection of SOD, Nrf2 and p-Nrf2 protein. Total cell lysates were prepared from pRISMCs treated with TEE or Lop+TEE, as described in Materials and Methods. The expression level of three proteins were detected with specific antibodies and quantified using an imaging densitometer. Three samples were assayed in duplicate by DCF-DA staining assay and western blotting. The data are reported as the mean +- SD. *, p < 0.05 compared with the No treated group. #, p < 0.05 compared with the Lop+Vehicle treated group. Abbreviations: ROS, Reactive oxygen species; DCF-DA, 2',7'-dichlorofluorescein diacetate; SOD, Superoxide dismutase; pRISMCs, primary smooth muscle of rat intestine cells.
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- Fig 3 Antioxidant activity of TEE in transverse colon of Lop+TEE treated constipation rats. (A) Detection of SOD proteins. The expression level of SOD was measured by Western blot analysis using specific antibodies. The relative level of this protein in each group was calculated based on the intensity of actin, after calculating the intensity of each band. (B) Determination of SOD activity. The SOD activity level was measured in homogenates of transverse colon tissue collected from each subset group, as described in Materials and Methods. One SOD unit is defined as the amount of enzyme in 20 muL of the sample solution that inhibits the reduction reaction of water-soluble tetrazolium salt-1 (WST-1) with superoxide anion by 50%. (C) Detection of SOD mRNA. The levels of SOD transcripts in the total messenger RNA (mRNA) of brain were measured by RT-qPCR analyses using specific primers. The mRNA level of SOD gene was calculated, based on the intensity of actin as an endogenous control. (D) Detection of Nrf2, and p-Nrf2 protein. The expression level of the two proteins was measured by Western blot analysis using specific antibodies. Subsequently, the phosphorylation level of a specific protein was calculated by dividing the level of phosphorylated proteins by the level of total proteins. (E) Determination of CAT activity. The CAT activity was measured in homogenates of transverse colon collected from each subset group, as described in Materials and Methods. Catalase 1 unit is defin
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- Fig. 3 Nrf2 activation following ocular hypertension. A) Quantification of Nrf2 mRNA. B) Representative western blots of NRF2. C) Quantification of NRF2 Western blot after normalization to beta-actin. D) Representative western blots for total and phosphorylated NRF2. E) Quantification of pNRF2 after normalization to total NRF2, ****p < 0.00001. F) Representative KEAP-1 western blots with or without immunoprecipitation of NRF2. G, H) Quantification of total KEAP-1. I) Quantification of KEAP-1 that immunoprecipitated with NRF2, *p < 0.05, **p < 0.001, ****p < 0.00001. Fig. 3
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- Fig. 4 AKT-dependent NRF2 phosphorylation. A) Representative western blots of AKT and pAKT B) Quantification of pAKT to total AKT, *p < 0.05. C) Experiment timeline. D) Representative Western blot of phosphorylated NRF2 to total NRF2. E) Quantification of pNRF2 to total NRF2, ***p < 0.0001. F) Quantification of antioxidant gene transcription shown as fold change over saline, **p < 0.001. G) Representative western blots for beta-actin, PRDX6, SOD3 and GPX1. H-J) Quantification of PRDX6, GPX1 and SOD3, respectively, after normalization to beta-actin, **p < 0.001, ***p < 0.0001. Fig. 4
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- Fig. 1 Immunostaining of phospho-aSyn (S129) and phospho-NRF2 (S40) in post-mortem control and PD midbrain sections. A-D Representative IHC images showing phospho-aSyn (S129) and phospho-NRF2 (S40) immunostaining in substantia nigra (SN) and periaqueductal grey (PAG) of two controls ( A - B ) and four PD cases ( C , D ). Red arrows in the 20 x magnified views point to cells with predominant nuclear localization of phospho-NRF2 (scale bar = 100 um). Also see Additional file 1 : Figure S1 showing panoramic and magnified views from a control and a PD case and Additional file 1 : Table S1. Primary antibodies: p-aSyn (S129)-81A (in A , C ) and p-NRF2 (S40)-EP1809Y (in B , D ). E, F Semi-quantitative analyses of p-aSyn (pS129; in E) and p-NRF2 (pS40; in F ) immunopositive cells in the indicated regions of control and PD midbrain sections. Individual data points represent immunopositive cell counts/mm 2 /region in each section, with controls being depicted as black triangles and PD as the black squares. ( SN substantia nigra; PAG periaqueductal grey matter; One-way ANOVA: ns, not significant; *** p < 0.0001)
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- Fig. 4 Immunostaining of phospho-NRF2 (S40) in the brain regions of PFF aSyn injected M83 +/+ mice. A Representative IHC images showing phospho-NRF2 (S40) immunostaining with distinct nuclear staining in brainstem regions (red arrows). Also, notice the predominantly cytoplasmic staining in cerebellar lobules (purkinje cells), motor cortex and corpus striatum-yellow arrows (10 x low magnification views and 40 x magnified views in the insets; scale bar = 200 um; Aq. in the image showing PAG, cerebral aqueduct; 4v. in the image showing vestibular nuclei image, 4th ventricle). Bregma co-ordinates for the brain regions were determined, according to Paxinos and Franklin: (Bregma, - 3.63 mm) midbrain at the level of superior colliculi showing PAG, red nucleus and tegmentum; (Bregma, - 5.79) pontocerebellar junction showing cerebellar nuclei, cerebellar lobules ( cb1-5 ), vestibular nuclei and pontine gigantocellualr nuclei (Gi); and (Bregma, 0.73 mm) forebrain showing motor cortex (M1 and M2) and corpus striatum. Also see Additional file 1 : Fig. S5A (PBS injected cohort) and Additional file 1 : Fig. S6B, C (additional high resolution data from the PFF and PBS cohorts). Primary antibody in A : p-NRF2 (S40)-PA5-67520. B Semi-quantitative analyses of p-NRF2 (pS40) immunopositive cells in the indicated brain regions of PBS or PFF injected M83 +/+ mice. Individual data points represent p-NRF2 (pS40) cell counts/mm 2 /region in each animal of the respective cohort, with PBS cohort being