33-1520

antibody from Invitrogen Antibodies

Targeting: OCLN PPP1R115

Provider product page for 33-1520

Western blot

ELISA

Other assay

Antibody data

Antibody Data
Antigen structure
References [9]
Comments [0]
Validations
Western blot [4]
Other assay [16]

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Product number: 33-1520 - Provider product page
Provider: Invitrogen Antibodies
Product name: Occludin Monoclonal Antibody (OC-3F10), HRP
Antibody type: Monoclonal
Antigen: Other
Reactivity: Human, Mouse, Rat, Canine
Host: Mouse
Conjugate: Horseradish Peroxidase
Isotype: IgG
Antibody clone number: OC-3F10
Vial size: 100 µg
Concentration: 1 mg/mL
Storage: 4° C

OCLN protein structure - 33-1520 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Occludin was performed by loading 10 µg of CaCO2 lysate per well onto a 4-12% Bis-Tris polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with an Occludin purified monoclonal antibody (Product # 33-1500) at a dilution of 1 µg/mL, 0.5 µg/mL, and 0.25 µg/mL for 1 hour at room temperature on a rocking platform and washed in TBS-0.1% Tween-20. Goat Anti-Mouse IGG (H+L) HRP Conjugated secondary antibody (Product # 31430) was used at a concentration of 1:20,000 and incubated for 30 minutes on a rocking platform. Super Signal West Dura substrate (Product # 34076) was incubated for 5 minutes on a rocking platform. Detection was performed using the GBox. The 5th isoform of Occludin was detected at ~23 kDa.
Conjugate: Horseradish Peroxidase

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockout of Occludin was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR777079_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of Occludin was performed by loading 30 µg of Caco-2 Cas9 (Lane 1) andCaco-2 Occludin KO (Lane 2) membrane enriched extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein 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 Anti-Occludin Monoclonal Antibody-HRP (OC-3F10) (Product # 33-1520, 1:500 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to Occludin.
Conjugate: Horseradish Peroxidase

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of Occludin was achieved by transfecting Caco-2 with Occludin specific siRNAs (Silencer® select Product # s9812, s9814). Western blot analysis (Fig. a) was performed using whole cell extracts from the Occludin knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Occludin Monoclonal Antibody (OC-3F10), HRP (Product # 33-1520, 2µg/ml). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Occludin.
Conjugate: Horseradish Peroxidase

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of Caco-2 (Lane 1) and Caco-2 treated with IL 1 beta (10 ng/ml for 48 hr) (Lane 2). The blot was probed with Anti-Occludin Monoclonal Antibody (OC-3F10), HRP (Product # 33-1520, 2 µg/ml). A 54, 30 kDa bands corresponding to Occludin was observed in the cell line tested and decreased upon treatment.
Conjugate: Horseradish Peroxidase

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL
Conjugate: Horseradish Peroxidase

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 HSD increases cerebral microvascular permeability. ( A ) Representative images of the mice cerebral cortical microvessels and dye leakage at 10 min after injection of Rhodamine B isothiocyanate-dextran (n=3 per group). ( B ) Evans blue leakage analyzed in rats fed with selected diets for 180 days (n=3 per group). ( C ) ZO-1 (green) co-stained with CD31 (red), a microvascular endothelia marker, and DAPI (blue; n=6 per group) in the rats brain slices. ( D , E ) Expression of Occludin, Claudin-5 and ZO-1 in the brain tissues from rats analyzed by immunoblotting. ( F ) Systolic blood pressure (SBP) of rats fed with NSD or HSD for 180 days. * P
Conjugate: Horseradish Peroxidase

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL
Conjugate: Horseradish Peroxidase

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL
Conjugate: Horseradish Peroxidase

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL
Conjugate: Horseradish Peroxidase

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 2 Effects of TNFalpha, IL-1beta and LPS on occludin band-shift in hCMEC/D3 cells. Cells were treated with or without TNFalpha (10 ng/mL) (A), IL-1beta (10 ng/mL)(B) and LPS (100 ng/mL) (C) for 15, 30 min and 1, 2, 4, 6 hours. Cell lysates were collected and occludin and beta-actin expression patterns were analyzed by immunoblotting assay as described in text. The density of upper band versus total upper and lower and normalized with beta-actin was determined by measuring protein intensity as PIupper/PItotal/PIbeta-actin. Results are mean +- SD from 4 or more experiments and data are analyzed by one-way ANOVA followed by Bonferroni post-tests (***P
Conjugate: Horseradish Peroxidase

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 4 TNFalpha-mediated occludin band-shift and phosphorylation of ERK1/2 and p38MAPK in hCMEC/D3 cells. Cells were treated with or without TNFalpha (10 ng/mL) at 5, 10, 15, 30, 60 min (A). Cell lysates were collected and occludin, P- ERK, T-ERK, P-P38, T-P38 and beta-actin expression pattern was analyzed by immunoblotting assay. Quantification of the proportion of occludin upper band were determined through PI upper /PI total /PI beta-actin and then normalized to control (B). phospho-ERK1/2 (C) and phospho-p38MAPK (D) were similarly quantified and plotted. Results are mean +- SD from 3 or more experiments and data are analyzed by one-way ANOVA followed by Bonferroni post-tests (**P
Conjugate: Horseradish Peroxidase

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 5 Effects of p-ERK1/2 and p-p38MAPK inhibitors on occludin band-shift in CMEC/D3 cells. Cells were pretreated with or without 10 muM U0126 and 10 muM SB202190 for 15 min and then stimulated with or without 10 ng/mL TNFalpha for 15, 30, 60 min (A and C). (B) Testing the effects of inhibitors alone. Cell lysates were collected and occludin, P- ERK, T-ERK, P-p38, T-p38 and beta-actin expression pattern was analyzed by immunoblotting assay. Quantification of the proportions of occludin upper band at 15 min of TNFalpha and with inhibitors (blots enclosed in brackets) were determined through PI upper /PI total /PI beta-actin and then normalized to control. Results are mean +- SD from 4 or more experiments. Two-way ANOVA revealed significant main effects of TNFalpha and the inhibitors and a significant interaction (p
Conjugate: Horseradish Peroxidase

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 6 Effects of TNFalpha on tyrosine and threonine phosphorylation of occludin. hCMEC/D3 cells were treated with or without 10 ng/mL TNFalpha for 15 min. Cell lysates were mixed with anti-tyrosine or anti-threonine antibody conjugated with protein A beads and then phosphorylated proteins in cell lysates were pulled down. Occludin and beta-actin expression pattern was analyzed by immunoblotting assay.
Conjugate: Horseradish Peroxidase

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 7 Effects of TNFalpha, IL-1beta and LPS on occludin expression and cell viability. (A) hCMEC/D3 cells were treated with or without TNFalpha (10 ng/mL), IL-1beta (10 ng/mL) and LPS (100 ng/mL) for 24 hours. Cell lysates were collected and occludin and beta-actin expression pattern was analyzed by immunoblotting assay. Quantification of the protein band intensity was determined through PI total /PI beta-actin and then normalized to control. Results are mean +- SD from 4 or more experiments and data are analyzed by one-way ANOVA followed by Bonferroni post-tests (***P
Conjugate: Horseradish Peroxidase

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 8 Effects of p-ERK1/2 and p-p38MAPK inhibitors on TNFalpha-induced occludin expression in hCMEC/D3 cells. (A) Initial testing for cell viability using WST-1 assay indicated toxicity of cells upon incubation (24h) with TNFalpha (10 ng/mL) in the presence of SB202190 at 10 muM but not at 2 muM. (B) Testing ability of U0126 (2 muM) and SB202190 (2 muM) to ameliorate the decrease in occludin expression upon exposure of TNFalpha for 24 h. Cell lysates were collected and occludin and beta-actin expression pattern was analyzed by immunoblotting assay. Quantification of protein band intensity was determined through PItotal/PIbeta-actin and then normalized to control. Results are mean +- SD from 5 or more experiments. Two-way ANOVA revealed a significant main effect of TNFalpha (p
Conjugate: Horseradish Peroxidase

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 HSD increases cerebral microvascular permeability. ( A ) Representative images of the mice cerebral cortical microvessels and dye leakage at 10 min after injection of Rhodamine B isothiocyanate-dextran (n=3 per group). ( B ) Evans blue leakage analyzed in rats fed with selected diets for 180 days (n=3 per group). ( C ) ZO-1 (green) co-stained with CD31 (red), a microvascular endothelia marker, and DAPI (blue; n=6 per group) in the rats brain slices. ( D , E ) Expression of Occludin, Claudin-5 and ZO-1 in the brain tissues from rats analyzed by immunoblotting. ( F ) Systolic blood pressure (SBP) of rats fed with NSD or HSD for 180 days. * P
Conjugate: Horseradish Peroxidase

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 Astrocyte-derived VEGF mediates HS-induced BBB breakdown. ( A ) Representative double immunofluorescence staining of ZO-1 + bEnd.3 endothelium. Endothelial cells were cultured alone, or co-cultured with primary rats' astrocytes, and treated with NS, HS, conditioned medium, or VEGF neutralizing antibody (NA). ( B , C ) Western blotting analysis of Occludin, Claudin-5, and ZO-1 in endothelial cells. ( D ) Permeability of tight junctions measured using NaF; * P
Conjugate: Horseradish Peroxidase

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6 Increased cerebral microvascular permeability in VEGF hi/+ mice. ( A ) Schematic diagram of VEGF hi/+ mice generation. ( B ) Immunoblotting showing VEGF, Occludin, Claudin-5 and ZO-1expression in VEGF hi/+ and wildtype mice. ( C ) Cerebral cortical micro-vessel and dye leakage at 5 and 10 min after injection of Rhodamine B isothiocyanate-dextran; n=5, * P
Conjugate: Horseradish Peroxidase

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 7 Blocking VEGF attenuates disruption of tight junctions induced by HSD. ( A ) Representative images showing double immunofluorescence staining of ZO-1 in green and DAPI in the brain specimens of rats. ( B , C ) Expression of Occludin, Claudin-5 and ZO-1 determined by western blotting; n=5, * P
Conjugate: Horseradish Peroxidase

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig 3 Effects of TNFalpha, IL-1beta and LPS on p-ERK1/2 and p-p38MAPK expression in hCMEC/D3 cells. Cells were treated with or without TNFalpha (A, B, C), IL-1beta (D) and LPS (E) for 15, 30 min and 1, 2, 4, 6 hours. Cell lysates were collected and phospho-ERK1/2 (P- ERK), total ERK1/2 (T-ERK), phospho-p38MAPK (P-P38), total p38MAPK (T-P38) and beta-actin expression pattern were analyzed by immunoblotting assay. Quantification of phospho-proteins was determined through assay of PIphospho/PItotal/PIbeta-actin and then normalized to control. Results of (B) and (C) are mean +- SD from 4 or more experiments and data are analyzed by one-way ANOVA followed by Bonferroni post-tests **P
Conjugate: Horseradish Peroxidase