- M421B - Invitrogen Antibodies IL1B antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-IL-1 beta Monoclonal Antibody (ILB1-H67) (Product # M421B) and a 35kDa band corresponding to Interleukin-1 beta was observed across in THP-1, upon treatment with Lipopolysaccharide. Whole cell extracts (30 µg lysate) of THP-1 (Lane 1), THP-1 (Lipopolysaccharide (1 µg/mL), 18 hours) (Lane 2), RAW 264.7 (Lane 3), BeWo (Lane 4) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0342BOX). 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 µg/mL) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

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Experimental details: Sandwich ELISA analysis of human IL-1b was performed using a Human IL-1b Colorimetric ELISA Kit (Product # EH2IL1B) by loading 50 µL per well of Human IL-1b Recombinant Protein (Product # SIL1B) in quadruplicate at 400, 160, 64, 25.6, 10.24 and 0 pg/mL across a 5 µg/mL mouse anti-human IL-1b (Product # M421B) pre-coated plate and incubating for 3 hours at room temperature. The plate was washed, then incubated with 50 µL per well of a biotinylated-mouse anti-human IL-1b (Product # M420BB) in quadruplicate at 0.07 µg/mL for 3 hours at room temperature. The plate was washed and incubated with 100 µL per well of Ultra Streptavidin-HRP (Product # N504) in all test wells at 1:3000 for 30 minutes at room temperature. Detection was performed using 1-Step Ultra TMB Substrate (Product # 34028) for 30 minutes at room temperature in the dark. The plate was then stopped with 0.16M sulfuric acid. Absorbances were read on a spectrophotometer at 450-550 nm.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Ovine 30-50 years old. Production of cytokines by purified PBMC of healthy donors from 30-50 years old group (ovine).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 8 Equine 30-50 years old. Production of cytokines by purified PBMC of healthy donors from 30-50 years old group (equine).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 9 Equine 50-70 years old. Production of cytokines by purified PBMC of healthy donors from 50-70 years old group (equine).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Inflammatory cytokines. ( A ) Immunohistochemical staining for IL-1beta and IL-18 on kidney sections. ( B ) Western blot and quantification of IL-1beta and IL-18, n = 3 for each group. ( C ) ELISA quantification of serum NLRP3, IL-6 and TNF-alpha, n = 5 for each group. Significant difference a p < 0.05 relative to untreated control; b p < 0.05 relative to AKI; c p < 0.05 relative to AKI + EVs; d p < 0.05 relative to AKI + EVs + pFUS.

Supportive validation

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Experimental details: 1 THP-1 monocytes but not Bac1 macrophages release large amounts of mIL-1beta in response to LPS stimulation. THP-1 or Bac-1 cells were split into six-well dishes at 1 x 10 6 /well. The cells were treated for 16 h with 500 ng/ml LPS, and the media was collected and centrifuged to remove cell debris. mIL-1beta released into the media was then quantified by ELISA. Inset: To control for production of pro-IL-1beta, the cells were lysed in SDS-PAGE buffer, and the lysates were subjected to Western blot analysis for pro-IL-1beta.

Supportive validation

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Experimental details: Fig. 2 Epithelial inflammatory cell infiltration, and elevated pro-inflammatory cytokine expression induced by burn injury. Ileal sections were stained with hematoxylin and eosin (H&E), and extensive microscopic inflammatory cell infiltration in burn group compared with the sham group was observed ( a , b , 200x). Cytokine protein levels in ileum of both groups were determined by Western blotting ( c ), and a summary of cytokines blots were presented as the ratio of cytokines:beta-actin densities ( d ). Cytokine protein levels in serum of both groups were determined by enzyme-linked immunosorbent assay (ELISA), and normalized to pg/ml of total serum volume ( e ). n = 5 per group. * p < 0.05, ** p < 0.01, *** p < 0.001. IL Interleukin, TNF-alpha Tumor necrosis factor-alpha

Supportive validation

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Experimental details: Fig. 3 Interleukin (IL)-17A neutralization attenuated intestinal pathological changes and zonula occludens-1 (ZO-1) alteration, suppressed intestinal permeability elevation, and inhibited increases of cytokines. Ileal sections were stained with hematoxylin and eosin (H&E) ( a , b , c 200x), and mucosa lensions were scored in each group( d ). Representative immunofluorescent images depicting membrane localization of ZO-1 in sham group ( e - g ), burn group ( h - j ), and burn+anti-IL-17A antibody group ( l - m ), red staining indicates ZO-1, and blue staining indicates nuclei (400x). Intestinal permeability was measured by fluorescein isothiocyanate (FITC)-dextran levels and is expressed as optical density (OD) value (mean +- standard error of the mean (SEM)) in per group ( n ). Cytokine protein levels in ileum of each group were determined by Western blotting ( o ), and summary of cytokines blots is presented as the ratio of cytokines: beta-actin densities ( p ). Cytokine protein levels in serum of each group were determined by enzyme-linked immunosorbent assay (ELISA), and normalized to pg/ml of total serum volume ( q ). n = 5 per group. * p < 0.05, ** p < 0.01, *** p < 0.001. DAPI 4',6-diamidino-2-phenylindole

Supportive validation

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Experimental details: 4 Cleaved IL-1beta selectively induces group 1 CD1 protein expression. (A, B) Monocytes were treated with tri-acyl-CSK4 at the indicated concentrations for 24 h and the amount of GM-CSF (A) and IL-1beta (B) in the supernatant measured by capture ELISA. (C) Monocytes were treated with the recombinant mature IL-1beta at the concentrations indicated and the percentage of cells expressing CD1a, CD1b, CD1c or CD1d, assessed by FACS analysis. The data are expressed in a normalize form so that the channel with the most cells is designated 100% of maximal (MAX). (D) Monocytes were cultured for 24 h with the indicated dose of triacyl-SK 4 and ATP (1 mM), either alone or in combination. Total lysates were prepared and western blotted for IL-beta (left panel) or CD1a-positive cells were assessed by FACS (right panel). Data shown are representative of more than three experiments.

M421B

Antibody data