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.).
Hmga2
Manufactured by Abcam
Sourced in United States
HMGA2 is a protein-coding gene that plays a role in transcriptional regulation. It is involved in cell growth and differentiation processes. The HMGA2 protein acts as an architectural transcription factor, modulating chromatin structure and regulating gene expression.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Abcam (3 mentions)
Histone h3, by Abcam (2 mentions)
Hmgb2, by Abcam (1 mentions)
Quantity one 4, by Bio-Rad (1 mentions)
Hmgb1, by Cell Signaling Technology (1 mentions)
Iblot stack, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase hrp, by GE Healthcare (1 mentions)
Igf2bp1, by Proteintech (1 mentions)
Hsp90, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Tsg101, by Abcam (1 mentions)
C myc, by Abcam (1 mentions)
Lin28a, by Abcam (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Snail, by Abcam (1 mentions)
Anti β actin peroxidase antibody, by Merck Group (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Bcl 2, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
13 protocols using hmga2
1
Western Blot Analysis of HMGA Proteins
2
Western Blot Analysis of Key Proteins
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Whole-cell lysates were prepared in RIPA buffer containing protease inhibitors and were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto nitrocellulose membranes (Invitrogen iBlot Stack). Incubations with primary antibodies recognizing KRAS (Abcam), IGF2BP1 (Proteintech), HMGA2 (Abcam), P21 (Millipore), TP53, GAPDH or HSP90 (Santa Cruz Biotech), were followed by incubations with the appropriate secondary antibodies conjugated with horseradish peroxidase (HRP; GE Healthcare). Signals were developed using Enhanced Chemiluminescence (ECL).
3
Western Blot Characterization of Exosomes
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Cells and exosomes were lysed with RIPA lysis buffer (Sigma, USA) supplemented with a 1× protease inhibitor mixture (Thermo, USA). Protein concentrations were determined with BCA assays. The experimental procedures were performed according to standard protocols. Briefly, the proteins were separated on SDS-polyacrylamide gels and then blotted onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% milk for 1 h, incubated with specific primary antibodies overnight at 4°C and horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature, and then subjected to chemiluminescence detection. Antibodies against PDGFR-β, Lin28B, HMGA2, TSG101, Alix and c-MYC were obtained from Abcam (USA). Antibodies against PDGFB were obtained from Sigma-Aldrich (Germany). Antibodies against β-actin, α-SMA and CD63 and secondary antibodies were obtained from Huabio (Hangzhou, China). Densitometry analyses of Western blots were performed with the Image-J software (NIH, USA), and the β-actin was used as the internal control.
4
Quantitative Protein Analysis via Western Blotting
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Western blot was performed as described previously (13 (link)). Briefly, separated proteins in SDS-PAGE were
transferred onto polyvinylidene fluoride membranes and were sequentially
immune-reacted with specific primary antibodies and secondary antibodies.
Antibody-conjunct proteins were quantified using SuperSignal™ West Pico
Chemiluminescent Substrate (Thermo Scientific, USA). The primary antibodies
cleaved caspase-3 (Cell Signaling Technology (CST, USA)), Bcl-2 (CST), Bax
(CST), Lin28a (CST), Sp-1 (CST), HMGA2 (CST), K-ras (CST), Snail (CST), and
Zcchc11 (Abcam, UK) were applied after the membranes were blocked in either 5%
milk or 5% BSA. Anti-β-actin-peroxidase antibody was obtained from Sigma-Aldrich
(USA) and used as an internal reference. The protein content was analyzed using
Image Lab 5.1 (Bio-Rad, USA).
transferred onto polyvinylidene fluoride membranes and were sequentially
immune-reacted with specific primary antibodies and secondary antibodies.
Antibody-conjunct proteins were quantified using SuperSignal™ West Pico
Chemiluminescent Substrate (Thermo Scientific, USA). The primary antibodies
cleaved caspase-3 (Cell Signaling Technology (CST, USA)), Bcl-2 (CST), Bax
(CST), Lin28a (CST), Sp-1 (CST), HMGA2 (CST), K-ras (CST), Snail (CST), and
Zcchc11 (Abcam, UK) were applied after the membranes were blocked in either 5%
milk or 5% BSA. Anti-β-actin-peroxidase antibody was obtained from Sigma-Aldrich
(USA) and used as an internal reference. The protein content was analyzed using
Image Lab 5.1 (Bio-Rad, USA).
5
Protein Extraction and Immunoblotting
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Cells (1–2 × 106) were collected, washed with PBS, resuspended in 50 mL of PBS with protease inhibitor (Halt) and then mixed with 50 mL of 2× Laemmli buffer with 5% β-mercaptoethanol. Samples were boiled for 10 min and then loaded onto 4%–12% NuPage gels. Antibodies were against HMGA2 (Abcam, Cambridge, MA, USA) and Actin (SCBT, Dallas, TX, USA).
6
Erythroblast Nuclear and Cytoplasmic Protein Extraction
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Nuclear and cytoplasmic extracts from culture day 14 erythroblasts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction kit (Pierce Biotechnology, Rockford, IL) as previously described [11 (link)]. Western blot protocols and conditions were performed as previously described [11 (link)]. Blots were probed with antibodies against CA1 (Abcam, Cambridge, MA), GCNT2 (Santa Cruz Biotechnology, Dallas, TX), BCL11A (Abcam), HMGA2 (GeneTex, Irvine, CA), ZBTB7A (Abcam), KLF1 (Abcam) and SOX6 (Santa Cruz Biotechnology). Histone H3, Lamin B1 or Beta-Actin (all from Abcam) were used as loading controls.
7
Western Blot Analysis of Pluripotency Markers
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Western blot analysis was performed as described previously.47 (link) The following primary antibodies were used: macroH2A1 (ab-37264, Abcam, Cambridge, MA, USA), Histone H3 (ab1791, Abcam), Histone H2A (Abcam), OCT4 (611203, BD Biosciences, San Jose, CA, USA), SOX2 (SC-20088, Santa Cruz, Santa Cruz, CA, USA), C-Myc (SC-40, Santa Cruz), KLF4 (ab75486, Abcam), HMGA2 (ab52039, Abcam), AURKA (610939, BD Biosciences), H-RAS (18295-1-ap, Proteintech, Chicago, IL, USA), Lin28B (#4196, Cell Signaling, Beverly, MA, USA), CHK1 (#2360, Cell Signaling), pCHK1 (#2348, Cell Signaling), CHK2 (#2662, Cell Signaling), pCHK2 (#2662, Cell Signaling) and β-actin (A5316, Sigma, St. Louis, MO, USA).
8
Western Blot Analysis of HMGA2 Expression
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For WB, all cells were collected and lysed in RIPA lysis buffer. Proteins were resolved on SDS/PAGE gels and transferred proteins to negative controls (NC) membranes, then incubated membrane in blocking buffer for 1 h and incubated primary antibody overnight. Next day, washed membrane in PBS for three times and incubated second antibody for 1 h. The primary antibodies used were: HMGA2 (Abcam, ab97276), β-actin (Abcam, ab8226).
9
Immunohistochemical Profiling of Index Case
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Immunohistochemistry of the index case was performed on paraffin-embedded tissue sections using antibodies against pan-cytokeratin AE1-AE3 (monoclonal mouse antihuman pan-cytokeratin, clone AE1/AE3, M3515; Dako Corp., Carpinteria, CA), Epithelial membrane antigen (EMA; clone E29, M0613; Dako), MDM2 (clone IF2; Zymed Laboratories, San Francisco, CA), HMGA2 (clone AB52039; Abcam, Paris, France), Cyclin Dependent Kinase 4 (CDK4; clone AHZ 0202; Invitrogen, Waltham, MA), TLE1 (clone 1F5; Cell Marque Sigma-Aldrich, Darmstadt, Germany), INI1/BAF47/SMARCB1 (clone 25, Becton Dickinson, San Jose, CA) and H3K27me3 (Diagenode, Seraing, Belgium).
10
Immunoblotting of Cell Lysates
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The cells were lysed and analysed by immunoblotting using antibodies specific for the following proteins: HMGA2 (Abcam; dilution 1:1000); α-tubulin (dilution 1:4000), p-p38 (dilution 1:2000), p38 (dilution 1:6000), p-eIF2α (dilution 1:500), JNK (dilution 1:1000), CAMKII (dilution 1:2000), p-CAMKII (dilution 1:200) and p-β-catenin (Ser552; dilution 1:500) (Cell Signalling Technology); and β-catenin (Santa Cruz biotechnology; dilution 1:1000).
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