Cells were lysed in RIPA buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, and protease inhibitors) and briefly sonicated. Protein content was measured using the Coomassie (Bradford) protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA), and equal amounts of cell lysate were separated via SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (GE Healthcare Biosciences, Foster City, CA, USA). Membranes were immunoblotted with antibodies against HMGB2 (Abcam, Cambridge, UK), p16, p21, and β-actin (Santa Cruz Biotechnology, Dallas, TX, USA), and detected via chemiluminescence using ECL detection reagents.
Hmgb2
Manufactured by Abcam
Sourced in United Kingdom
HMGB2 is a high mobility group box protein that functions as a DNA-binding and architectural transcription factor. It is involved in the regulation of gene expression and plays a role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Coomassie bradford protein assay kit, by Thermo Fisher Scientific (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Hmga2, by Abcam (1 mentions)
Quantity one 4, by Bio-Rad (1 mentions)
Hmgb1, by Cell Signaling Technology (1 mentions)
β actin, by Abcam (1 mentions)
Total protein extraction buffer, by Beyotime (1 mentions)
Nf κb p65, by Abcam (1 mentions)
Hrp labeled goat anti rabbit igg, by Beyotime (1 mentions)
Cmpk1, by Abcam (1 mentions)
5 aza decitabine, by Selleck Chemicals (1 mentions)
Dnmt1, by Abcam (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti α tubulin, by Santa Cruz Biotechnology (1 mentions)
Anti p21, by Beyotime (1 mentions)
Anti p53, by Beyotime (1 mentions)
Anti myc, by Santa Cruz Biotechnology (1 mentions)
Lncrna smart silencer, by RiboBio (1 mentions)
Anti flag, by Cell Signaling Technology (1 mentions)
7 protocols using hmgb2
1
Immunoblot Analysis of Cell Signaling
2
Western Blot Analysis of HMGA Proteins
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Cell lysates were obtained using a lysis buffer containing phosphatase inhibitor and the protease inhibitor phenylmethanesulfonyl fluoride (100 mM; cat. no. KGP250; Nanjing KeyGen Biotech Co., Ltd.) and the protein concentration in the lysates determined by Bradford assay. A total of 100 µg lysate was separated by 10% SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane at 250 mA for 1 h. The PVDF membranes were blocked with 5% FBS for 1.5 h at 37°C and incubated overnight at 4°C with rabbit anti-mouse antibodies against high mobility group (HMG)A1 (1:10,000; cat. no. ab129153; Abcam), HMGA2 (1:1,000; cat. no. D1A7; Cell Signaling Technology, Inc.), HMGB1 (1:10,000; cat. no. ab79823; Abcam), HMGB2 (1:10,000; cat. no. ab124670; Abcam) and β-actin (1:1,000; cat. no. BM0627; Wuhan Boster Biological Technology, Ltd.). The PVDF membranes were washed three times with TBST (0.1% Tween) and incubated with goat anti-rabbit Immunoglobulin G-HRP (1:1,000; cat. no. BA1054; Wuhan Boster Biological Technology, Ltd.) for 1.5 h at 37°C, followed by the 3,3′-diaminobenzidine (EMD Millipore) method at room temperature for 15 sec. PVDF membranes were subjected to densitometry analysis (chemiDox.XRS+; Bio-Rad Laboratories, Inc.), then the image was analyzed using Quantity One 4.0 software (Bio-Rad Laboratories, Inc.).
3
Protein Extraction and Western Blot Analysis
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Total protein from the cells was extracted using a total protein extraction buffer (Beyotime, China). 10% sodium dodecyl sulfate polyacrylamide gel was used to isolate the proteins. After being transferred to nitrocellulose membrane, the band was blocked with 5% nonfat milk. The blots were incubated with primary antibodies and second antibodies, respectively. The antibodies and reagents were used as follows: HMGB2 (Abcam, Ab124670); JNK1/2 (CST, #9252); P-JNK1/2 (CST, #4668); Cleaved caspase3 (CST, #9661); Cleaved PARP (CST, #9545); NF-κB P65 (Abcam, Ab16502); GAPDH (CST, #5174); H3 (CST, #4499); HRP-labeled Goat Anti-Rabbit IgG (Beyotime, A0208). The results were used to visualize proteins by the enhanced chemiluminescence reagents (Tanon, China).
4
Molecular Mechanisms of Epigenetic Regulation
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HUVECs were transfected using the Effectence transfection reagent (Qiagen, Suzhou, Jiangsu, China), while HEK293T cells were transfected using the Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA). 5-aza (Decitabine), a potent inhibitor of DNA methylation, was from Selleck Chemicals (Shanghai, China). siRNAs were synthesized from Genepharma (Suzhou, China), the sequences of siRNAs are listed in S1 Table . LncRNA Smart Silencer was obtained from RiboBio (Guangzhou, China). Antibodies against KSHV LANA, HMGB2, CMPK1, DNMT1 and Dicer were from Abcam (Cambridge, MA, USA). Anti-Flag was obtained from Cell Signaling Technologies (Beijing, China). Anti-Myc, anti-α-Tubulin, and anti-GAPDH were from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-rabbit immunoglobulin G (IgG), anti-mouse IgG, anti-phosphorylated p53, anti-acetylated p53, anti-p53, and anti-p21 were purchased from Beyotime Institute of Biotechnology (Nantong, Jiangsu, China). Western-blotting analysis was conducted as previously described [81 (link), 82 (link)]. In this study, all Western blotting results were independently repeated at least three times unless otherwise stated.
5
Analyzing HMGB2, p53, and ATM Proteins
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Cells were lysed in RIPA buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, and protease inhibitors] and briefly sonicated. Protein content was measured using the Coomassie (Bradford) Protein Assay kit (Thermo Fisher Scientific, Inc., USA), and equal amounts of cell lysate were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad). Membranes were immunoblotted with antibodies against HMGB2 (Abcam, UK), p53, p21, β-actin (Santa Cruz Biotechnology, USA), and ATM (Epitomics, USA), and detected by chemiluminescence using ECL detection reagents.
6
Quantification of HMGB2 and β-actin by Western Blotting
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HMGB2 and β-actin were quantified by Western blotting. Briefly, DAKIKI cells were collected and lysed, after which the proteins were extracted. Protein samples were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis for 50 min and transferred to polyvinylidene difluoride membranes for 3 h. Bands were excised according to the molecular weight of the target protein and blocked (5% BSA) for 2 h at room temperature. Primary antibodies diluted with blocking solution were added and incubated overnight at 4 °C or room temperature for 2 h. The samples were washed three times with TBST (10 min), incubated with secondary antibodies (HMGA1: Abcam, England; HMGB2: Abcam, England; β-actin: Servicebio, China) for 1 h at room temperature, and then washed three times at room temperature (10 min). The membrane was immersed in a chemiluminescent substrate (Solarbio, China) for 2 min and visualized in a Bio-Rad gel imaging system (USA).
7
Fetal Pig Heart Development Immunohistochemistry
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A pig embryo at 27 dpf was fixed overnight in 10% neutral formalin-fixed solution at room temperature. Thin 5-μm TV and LV paraffin-embedded sections were collected for immunofluorescence staining. Cell nuclei were stained using DAPI (Catalog No. 62247, Life Technologies, Carlsbad, CA).
Primary antibodies used were: MATN1 (Catalog No. orb94279, Biorbyt, Cambridgeshire, UK), HMGB2 (Catalog No. ab67282, Abcam, Cambridgeshire, UK), COL1A1 (Catalog No. ab34710, Abcam), CD34 (Catalog No. orb348961, Biorbyt), CD93 (Catalog No. ab198854, Abcam), and CD248 (Catalog No. sc-377221, Santa Cruz Biotechnology, Delaware, CA). The secondary antibody used was anti-rabbit IgG (Catalog No. ZDR-5003, ZSGB-BIO, Beijing, China).
Primary antibodies used were: MATN1 (Catalog No. orb94279, Biorbyt, Cambridgeshire, UK), HMGB2 (Catalog No. ab67282, Abcam, Cambridgeshire, UK), COL1A1 (Catalog No. ab34710, Abcam), CD34 (Catalog No. orb348961, Biorbyt), CD93 (Catalog No. ab198854, Abcam), and CD248 (Catalog No. sc-377221, Santa Cruz Biotechnology, Delaware, CA). The secondary antibody used was anti-rabbit IgG (Catalog No. ZDR-5003, ZSGB-BIO, Beijing, China).
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