Human HMGB1 knockout HeLa cell line (ab255395)
Overview
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Product name
Human HMGB1 knockout HeLa cell line -
Parental Cell Line
HeLa -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, Homozygous: 1 bp deletion in exon 2 -
Passage number
<20 -
Knockout validation
Sanger Sequencing -
Tested applications
Suitable for: WBmore details -
Biosafety level
2 -
General notes
Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images.
Recommended control: Human wild-type HeLa cell line (ab255928). Please note a wild-type cell line is not automatically included with a knockout cell line order, if required please add recommended wild-type cell line at no additional cost using the code WILDTYPE-TMTK1.
Cryopreservation cell medium: Cell Freezing Medium-DMSO Serum free media, contains 8.7% DMSO in MEM supplemented with methyl cellulose.
Culture medium: DMEM (High Glucose) + 10% FBS
Initial handling guidelines: Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.
1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.
2. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 mL represents minimum recommended dilution. 20 mL represents maximum recommended dilution.
3. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method. Based on cell count, seed cells in an appropriate cell culture flask at a density of 2x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules.
4. Incubate the culture at 37°C incubator with 5% CO2. Cultures should be monitored daily.Subculture guidelines:
- All seeding densities should be based on cell counts gained by established methods.
- A guide seeding density of 2x104 cells/cm2 is recommended.
- A partial media change 24 hours prior to subculture may be helpful to encourage growth, if required.
- Cells should be passaged when they have achieved 80-90% confluence.
This product is subject to limited use licenses from The Broad Institute and ERS Genomics Limited, and is developed with patented technology. For full details of the limited use licenses and relevant patents please refer to our limited use license and patent pages.
We will provide viable cells that proliferate on revival.
Properties
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Number of cells
1 x 106 cells/vial, 1 mL -
Adherent /Suspension
Adherent -
Tissue
Cervix -
Cell type
epithelial -
Disease
Adenocarcinoma -
Gender
Female -
STR Analysis
Amelogenin X D5S818: 11, 12 D13S317: 12, 13.3 D7S820: 8, 12 D16S539: 9, 10 vWA: 16, 18 TH01: 7 TPOX: 8,12 CSF1PO: 9, 10 -
Mycoplasma free
Yes -
Storage instructions
Shipped on Dry Ice. Store in liquid nitrogen. -
Storage buffer
Constituents: 8.7% Dimethylsulfoxide, 2% Cellulose, methyl ether -
Research areas
Target
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Function
Multifunctional redox sensitive protein with various roles in different cellular compartments. In the nucleus is one of the major chromatin-associated non-histone proteins and acts as a DNA chaperone involved in replication, transcription, chromatin remodeling, V(D)J recombination, DNA repair and genome stability. Proposed to be an universal biosensor for nucleic acids. Promotes host inflammatory response to sterile and infectious signals and is involved in the coordination and integration of innate and adaptive immune responses. In the cytoplasm functions as sensor and/or chaperone for immunogenic nucleic acids implicating the activation of TLR9-mediated immune responses, and mediates autophagy. Acts as danger associated molecular pattern (DAMP) molecule that amplifies immune responses during tissue injury. Released to the extracellular environment can bind DNA, nucleosomes, IL-1 beta, CXCL12, AGER isoform 2/sRAGE, lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and activates cells through engagement of multiple surface receptors. In the extracellular compartment fully reduced HMGB1 (released by necrosis) acts as a chemokine, disulfide HMGB1 (actively secreted) as a cytokine, and sulfonyl HMGB1 (released from apoptotic cells) promotes immunological tolerance (PubMed:23519706, PubMed:23446148, PubMed:23994764, PubMed:25048472). Has proangiogdenic activity (By similarity). May be involved in platelet activation (By similarity). Binds to phosphatidylserine and phosphatidylethanolamide (By similarity). Bound to RAGE mediates signaling for neuronal outgrowth (By similarity). May play a role in accumulation of expanded polyglutamine (polyQ) proteins such as huntingtin (HTT) or TBP (PubMed:23303669, PubMed:25549101).
Nuclear functions are attributed to fully reduced HGMB1. Associates with chromatin and binds DNA with a preference to non-canonical DNA structures such as single-stranded DNA, DNA-containing cruciforms or bent structures, supercoiled DNA and ZDNA. Can bent DNA and enhance DNA flexibility by looping thus providing a mechanism to promote activities on various gene promoters by enhancing transcription factor binding and/or bringing distant regulatory sequences into close proximity (PubMed:20123072). May have an enhancing role in nucleotide excision repair (NER) (By similarity). However, effects in NER using in vitro systems have been reported conflictingly (PubMed:19446504, PubMed:19360789). May be involved in mismatch repair (MMR) and base excision repair (BER) pathways (PubMed:15014079, PubMed:16143102, PubMed:17803946). May be involved in double strand break repair such as non-homologous end joining (NHEJ) (By similarity). Involved in V(D)J recombination by acting as a cofactor of the RAG complex: acts by stimulating cleavage and RAG protein binding at the 23 bp spacer of conserved recombination signal sequences (RSS) (By similarity). In vitro can displace histone H1 from highly bent DNA (By similarity). Can restructure the canonical nucleosome leading to relaxation of structural constraints for transcription factor-binding (By similarity). Enhances binding of sterol regulatory element-binding proteins (SREBPs) such as SREBF1 to their cognate DNA sequences and increases their transcriptional activities (By similarity). Facilitates binding of TP53 to DNA (PubMed:23063560). Proposed to be involved in mitochondrial quality control and autophagy in a transcription-dependent fashion implicating HSPB1; however, this function has been questioned (By similarity). Can modulate the activity of the telomerase complex and may be involved in telomere maintenance.
In the cytoplasm proposed to dissociate the BECN1:BCL2 complex via competitive interaction with BECN1 leading to autophagy activation (PubMed:20819940). Involved in oxidative stress-mediated autophagy (PubMed:21395369). Can protect BECN1 and ATG5 from calpain-mediated cleavage and thus proposed to control their proautophagic and proapoptotic functions and to regulate the extent and severity of inflammation-associated cellular injury (By similarity). In myeloid cells has a protective role against endotoxemia and bacterial infection by promoting autophagy (By similarity). Involved in endosomal translocation and activation of TLR9 in response to CpG-DNA in macrophages.
In the extracellular compartment (following either active secretion or passive release) involved in regulation of the inflammatory response. Fully reduced HGMB1 (which subsequently gets oxidized after release) in association with CXCL12 mediates the recruitment of inflammatory cells during the initial phase of tissue injury; the CXCL12:HMGB1 complex triggers CXCR4 homodimerization (PubMed:22370717). Induces the migration of monocyte-derived immature dendritic cells and seems to regulate adhesive and migratory functions of neutrophils implicating AGER/RAGE and ITGAM (By similarity). Can bind to various types of DNA and RNA including microbial unmethylated CpG-DNA to enhance the innate immune response to nucleic acids. Proposed to act in promiscuous DNA/RNA sensing which cooperates with subsequent discriminative sensing by specific pattern recognition receptors (By similarity). Promotes extracellular DNA-induced AIM2 inflammasome activation implicating AGER/RAGE (PubMed:24971542). Disulfide HMGB1 binds to transmembrane receptors, such as AGER/RAGE, TLR2, TLR4 and probably TREM1, thus activating their signal transduction pathways. Mediates the release of cytokines/chemokines such as TNF, IL-1, IL-6, IL-8, CCL2, CCL3, CCL4 and CXCL10 (PubMed:12765338, PubMed:18354232, PubMed:19264983, PubMed:20547845, PubMed:24474694). Promotes secretion of interferon-gamma by macrophage-stimulated natural killer (NK) cells in concert with other cytokines like IL-2 or IL-12 (PubMed:15607795). TLR4 is proposed to be the primary receptor promoting macrophage activation and signaling through TLR4 seems to implicate LY96/MD-2 (PubMed:20547845). In bacterial LPS- or LTA-mediated inflammatory responses binds to the endotoxins and transfers them to CD14 for signaling to the respective TLR4:LY96 and TLR2 complexes (PubMed:18354232, PubMed:21660935, PubMed:25660311). Contributes to tumor proliferation by association with ACER/RAGE (By similarity). Can bind to IL1-beta and signals through the IL1R1:IL1RAP receptor complex (PubMed:18250463). Binding to class A CpG activates cytokine production in plasmacytoid dendritic cells implicating TLR9, MYD88 and AGER/RAGE and can activate autoreactive B cells. Via HMGB1-containing chromatin immune complexes may also promote B cell responses to endogenous TLR9 ligands through a B-cell receptor (BCR)-dependent and ACER/RAGE-independent mechanism (By similarity). Inhibits phagocytosis of apoptotic cells by macrophages; the function is dependent on poly-ADP-ribosylation and involves binding to phosphatidylserine on the cell surface of apoptotic cells (By similarity). In adaptive immunity may be involved in enhancing immunity through activation of effector T cells and suppression of regulatory T (TReg) cells (PubMed:15944249, PubMed:22473704). In contrast, without implicating effector or regulatory T-cells, required for tumor infiltration and activation of T-cells expressing the lymphotoxin LTA:LTB heterotrimer thus promoting tumor malignant progression (By similarity). Also reported to limit proliferation of T-cells (By similarity). Released HMGB1:nucleosome complexes formed during apoptosis can signal through TLR2 to induce cytokine production (PubMed:19064698). Involved in induction of immunological tolerance by apoptotic cells; its pro-inflammatory activities when released by apoptotic cells are neutralized by reactive oxygen species (ROS)-dependent oxidation specifically on Cys-106 (PubMed:18631454). During macrophage activation by activated lymphocyte-derived self apoptotic DNA (ALD-DNA) promotes recruitment of ALD-DNA to endosomes. -
Tissue specificity
Ubiquituous. Expressed in platelets (PubMed:11154118). -
Sequence similarities
Belongs to the HMGB family.
Contains 2 HMG box DNA-binding domains. -
Domain
HMG box 2 mediates proinflammatory cytokine-stimulating activity and binding to TLR4 (PubMed:12765338, PubMed:20547845). However, not involved in mediating immunogenic activity in the context of apoptosis-induced immune tolerance (PubMed:24474694).
The acidic C-terminal domain forms a flexible structure which can reversibly interact intramolecularily with the HMG boxes and modulate binding to DNA and other proteins (PubMed:23063560). -
Post-translational
modificationsPhosphorylated at serine residues. Phosphorylation in both NLS regions is required for cytoplasmic translocation followed by secretion (PubMed:17114460).
Acetylated on multiple sites upon stimulation with LPS (PubMed:22801494). Acetylation on lysine residues in the nuclear localization signals (NLS 1 and NLS 2) leads to cytoplasmic localization and subsequent secretion (By similarity). Acetylation on Lys-3 results in preferential binding to DNA ends and impairs DNA bending activity.
Reduction/oxidation of cysteine residues Cys-23, Cys-45 and Cys-106 and a possible intramolecular disulfide bond involving Cys-23 and Cys-45 give rise to different redox forms with specific functional activities in various cellular compartments: 1- fully reduced HMGB1 (HMGB1C23hC45hC106h), 2- disulfide HMGB1 (HMGB1C23-C45C106h) and 3- sulfonyl HMGB1 (HMGB1C23soC45soC106so).
Poly-ADP-ribosylated by PARP1 when secreted following stimulation with LPS.
In vitro cleavage by CASP1 is liberating a HMG box 1-containing peptide which may mediate immunogenic activity; the peptide antagonizes apoptosis-induced immune tolerance (PubMed:24474694). Can be proteolytically cleaved by a thrombin:thrombomodulin complex; reduces binding to heparin and proinflammatory activities. -
Cellular localization
Nucleus. Chromosome. Cytoplasm. Secreted. Cell membrane. Endosome. Endoplasmic reticulum-Golgi intermediate compartment. In basal state predominantly nuclear. Shuttles between the cytoplasm and the nucleus (PubMed:12231511, PubMed:17114460). Translocates from the nucleus to the cytoplasm upon autophagy stimulation (PubMed:20819940). Release from macrophages in the extracellular milieu requires the activation of NLRC4 or NLRP3 inflammasomes (By similarity). Passively released to the extracellular milieu from necrotic cells by diffusion, involving the fully reduced HGMB1 which subsequently gets oxidized (PubMed:19811284). Also released from apoptic cells (PubMed:16855214, PubMed:18631454). Active secretion from a variety of immune and non-immune cells such as macrophages, monocytes, neutrophils, dendritic cells and natural killer cells in response to various stimuli such as LPS and cytokines involves a nonconventional secretory process via secretory lysosomes (PubMed:12231511, PubMed:14532127, PubMed:15944249). Secreted by plasma cells in response to LPS (By similarity). Found on the surface of activated platelets (PubMed:11154118). - Information by UniProt
Associated products
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KO cell lysates
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab255395 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
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WB |
Use at an assay dependent concentration. Predicted molecular weight: 24 kDa.
Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images. |
Notes |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 24 kDa. Western blot data indicates that the CRISPR gene edit may have resulted in a truncation of the protein of interest. Please see data images. |
Images
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All lanes : Anti-HMGB1 antibody [EPR3506] (ab92310) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : HMGB1 knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 24 kDa
Observed band size: 30 kDa why is the actual band size different from the predicted?Lanes 1- 2: Merged signal (red and green). Green - ab92310 observed at 30 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
ab92310 was shown to react with HMGB1 in wild-type HeLa cells in western blot. Loss of signal at the expected size was observed when knockout cell line ab255395 (knockout cell lysate ab263782) was used. The band observed in lane 2 below 25kDa may represent truncated forms and cleaved fragments. Wild-type HeLa and HMGB1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab92310 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
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All lanes : Anti-HMGB1 antibody [EPR3507] (ab79823) at 1/10000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : HMGB1 knockout HeLa cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 24 kDa
Observed band size: 30 kDa why is the actual band size different from the predicted?Lanes 1- 2: Merged signal (red and green). Green - ab79823 observed at 30 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.
ab79823 was shown to react with HMGB1 in wild-type HeLa cells in western blot. Loss of signal at the expected size was observed when knockout cell line ab255395 (knockout cell lysate ab263782) was used. The band observed in lane 2 below 25kDa may represent truncated forms and cleaved fragments. Wild-type HeLa and HMGB1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab79823 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 10000 Dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
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Homozygous: 1 bp deletion in exon 2.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab255395 has not yet been referenced specifically in any publications.