- PA1-16926 - Invitrogen Antibodies HMGB1 antibody

Submitted references Pentoxifylline prevents epileptic seizure via modulating HMGB1/RAGE/TLR4 signalling pathway and improves memory in pentylenetetrazol kindling rats.

Badawi GA, Shokr MM, Zaki HF, Mohamed AF
Clinical and experimental pharmacology & physiology 2021 Aug;48(8):1111-1124

The Potential of Nanobody-Targeted Photodynamic Therapy to Trigger Immune Responses.

Beltrán Hernández I, Angelier ML, Del Buono D'Ondes T, Di Maggio A, Yu Y, Oliveira S
Cancers 2020 Apr 15;12(4)

A small bioactive glycoside inhibits epsilon toxin and prevents cell death.

Shivappagowdar A, Pati S, Narayana C, Ayana R, Kaushik H, Sah R, Garg S, Khanna A, Kumari J, Garg L, Sagar R, Singh S
Disease models & mechanisms 2019 Oct 10;12(10)

Expression Analysis of Autophagy Related Markers LC3B, p62 and HMGB1 Indicate an Autophagy-Independent Negative Prognostic Impact of High p62 Expression in Pulmonary Squamous Cell Carcinomas.

Langer R, Neppl C, Keller MD, Schmid RA, Tschan MP, Berezowska S
Cancers 2018 Aug 21;10(9)

The presence of LC3B puncta and HMGB1 expression in malignant cells correlate with the immune infiltrate in breast cancer.

Ladoire S, Enot D, Senovilla L, Ghiringhelli F, Poirier-Colame V, Chaba K, Semeraro M, Chaix M, Penault-Llorca F, Arnould L, Poillot ML, Arveux P, Delaloge S, Andre F, Zitvogel L, Kroemer G
Autophagy 2016 May 3;12(5):864-75

Biomarkers of immunogenic stress in metastases from melanoma patients: Correlations with the immune infiltrate.

Ladoire S, Senovilla L, Enot D, Ghiringhelli F, Poirier-Colame V, Chaba K, Erdag G, Schaefer JT, Deacon DH, Zitvogel L, Slingluff CL Jr, Kroemer G
Oncoimmunology 2016 Jun;5(6):e1160193

Combined evaluation of LC3B puncta and HMGB1 expression predicts residual risk of relapse after adjuvant chemotherapy in breast cancer.

Ladoire S, Penault-Llorca F, Senovilla L, Dalban C, Enot D, Locher C, Prada N, Poirier-Colame V, Chaba K, Arnould L, Ghiringhelli F, Fumoleau P, Spielmann M, Delaloge S, Poillot ML, Arveux P, Goubar A, Andre F, Zitvogel L, Kroemer G
Autophagy 2015;11(10):1878-90

Negative prognostic impact of regulatory T cell infiltration in surgically resected esophageal cancer post-radiochemotherapy.

Vacchelli E, Semeraro M, Enot DP, Chaba K, Poirier Colame V, Dartigues P, Perier A, Villa I, Rusakiewicz S, Gronnier C, Goéré D, Mariette C, Zitvogel L, Kroemer G
Oncotarget 2015 Aug 28;6(25):20840-50

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot detection of HMGB1 in whole cell HeLa lysate using Product # PA1-16926. ECL exposure, 1 min.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of HMGB1 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR784665_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of HMGB1 was performed by loading 30 µg of HeLa Wild Type (Lane 1), HeLa Cas9 (Lane 2) andHeLa HMGB1 KO (Lane 3) whole cell 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-HMGB1 Polyclonal Antibody (Product # PA1-16926, 1:1,000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:5,000 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 HMGB1. An uncharacterized band was observed in all the samples at ~50 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of HMGB1 in SHSY-5Y, MCF7, Neuro2A and HeLa. Samples were incubated in HMGB1 polyclonal antibody (Product # PA1-16926) using a dilution of 2 µg/mL followed by an anti-rabbit HRP secondary antibody. Separated on a 12% gel by SDS-PAGE, transferred to PVDF membrane and blocked in 5% non-fat milk in TBST. Detection: chemiluminescence.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of HMGB1 in 0.05 mg/mL Jurkat lysate. Samples were incubated in HMGB1 polyclonal antibody (Product # PA1-16926). This experiment was performed under reducing conditions using the 12-230 kDa separation system.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of HMGB1 was achieved by transfecting MCF7 with HMGB1 specific siRNAs (Silencer® select Product # s20255, s20254). Western blot analysis (Fig. a) was performed using Whole cell extracts from the HMGB1 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with HMGB1 Polyclonal Antibody (Product # PA1-16926, 1 µg/mL ) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000). 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 HMGB1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-HMGB1 Polyclonal Antibody (Product # PA1-16926) and a ~30kDa band corresponding to HMGB1 was observed across cell lines and tissues tested . Whole cell extracts (30 µg lysate) of RAW 264.7 (Lane 1), MCF7 (Lane 2), SH-SY5Y (Lane 3), THP-1 (Lane 4), PC-12 (Lane 5), HEK-293 (Lane 6), A-431 (Lane 7), Hep G2 (Lane 8), HeLa (Lane 9), Mouse Liver (Lane 10), Mouse Ovary (Lane 11) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:4000) 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: Immunofluorescence analysis of HMGB1 was performed using 70% confluent log phase SH-SY5Y cells. 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 HMGB1 Polyclonal Antibody (Product # PA1-16926) at 0.05 µg/mL 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), for 45 minutes at room temperature (Panel a: Blue). Nuclei (Panel b:Green) 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). Panel d represents the merged image showing Nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Detectionof HMGB1 (Green) in Hela cells using Product # PA1-16926. Nuclei (Blue) are counterstained with Hoecsht 33258.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of HMGB1 in mouse liver. Samples were incubated in HMGB1 polyclonal antibody (Product # PA1-16926).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FACS staining of NTERA-2 cells using Product # PA1-16926 at a 1:50 dilution detected using Dylight-488 conjugated goat anti-rabbit IgG secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry of HMGB1 in HeLa and a matched isotype control. Samples were incubated in HMGB1 polyclonal antibody (Product # PA1-16926) using a dilution of 1 µg/mL for 30 minutes at room temperature followed by a Rabbit IgG (H+L) Cross-Adsorbed secondary antibody. Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry of HMGB1 in RH-30 cells. Samples were incubated in HMGB1 polyclonal antibody (Product # PA1-16926) using a dilution of 10 µg/mL for 30 minutes at room temperature. Antibody (blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Chromatin Immunoprecipitation (ChIP) assay of endogenous HMGB1 protein using HMGB1 Antibody: ChIP was performed using HMGB1 Polyclonal Antibody (Product # PA1-16926, 5 µg) on sheared chromatin from Hep G2 cells using the MAGnify ChIP System kit (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR using primers binding to MIA-PR (promoter), c myc promoter, TNF alpha (active) and SAT alpha and SAT2 satellite repeats (Inactive). Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

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: NULL

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 1 Examples of immunohistochemical stainings; ( A ) LC3B low, ( B ) LC3B high, ( C ) p62 low (any cellular compartment), ( D ) p62 cytoplasmic low, ( E ) p62 cytoplasmic/dot like high, nuclear low, ( F ) p62 cytoplasmic/dot like low, nuclear high, ( G ) HMGB1 low, ( H ) HMGB1 high. Objective magnification, 40x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 4 FIGURE Effect of pentoxifylline (PTX) on pro-inflammatory parameters (IL-1beta, HMGb1, TLR4, RAGE, and NF-kappaB) in hippocampi of pentylenetetrazol (PTZ)-kindled rats. (A): IL-1beta, (B); HMGB1, (C); TLR4, (D); RAGE, (E); NF-kappaB. (B1); Representative Western blot analysis of hippocampal HMGB1, (B2); Quantification of the level of hippocampal HMGB1. (C1); Representative Western blot analysis of hippocampal TLR4, (C2); Quantification of the level of hippocampal TLR4. Data are expressed as mean +- SEM of six rats of each group; p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4. Glycoside-4 protects MDCK cells from Etx-triggered cell death. (A) MDCK cells were treated with Etx or in combination with glycoside-4, and the annexin V staining of exposed phosphatidyl serine on the outer surface and PI entry were observed. The toxin-treated cells showed annexin and PI (annexin + /PI + ) dual positivity, whereas glycoside-4-treated cells showed the absence of both the markers (annexin - /PI - ). Scale bars: 20 mum. (B) Bar graph depicts mean fluorescence intensity (MFI) for annexin and PI. Statistical significance (*** P

PA1-16926

Antibody data