Mouse colonic mucosa was collected by scraping proximal and distal regions as previously described.20 (link) Tumors were collected separately. Mouse epithelial cells were lysed in lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 1 mM EGTA pH 8.0, 0. 2 mM sodium orthovanadate, protease inhibitor cocktail). Immunoblotting was performed with primary antibodies: anti-AvrA (custom-made in Sub lab13 (link)), anti-β-catenin (BD, Transduction Laboratories), anti-Bmi 1 (Abcam), and anti-villin (Santa Cruz Biotechnology Inc.), anti-GSK-3β (BD), anti-phospho-GSK-3β (Ser9), anti-phospho-β-catenin (Ser552), anti-phospho-β-catenin (Ser33, Ser37, Thr41) and anti-phospho-Akt (T308) (Cell Signaling, Beverly, MA, USA), or anti-β-actin (Sigma-Aldrich) antibodies, and visualized by enhanced chemiluminescence.43 (link), 44 (link)
Anti bmi1
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-BMI1 is a laboratory reagent used to detect the presence of the BMI1 protein in biological samples. BMI1 is a component of the Polycomb Repressive Complex 1 (PRC1), which plays a role in the regulation of gene expression. Anti-BMI1 can be used in various research applications, such as Western blotting, immunohistochemistry, and flow cytometry, to study the expression and localization of the BMI1 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti oct4, by Abcam (4 mentions)
Anti nanog, by Abcam (3 mentions)
Anti p53, by Abcam (2 mentions)
Pcna antibody, by Santa Cruz Biotechnology (2 mentions)
β catenin, by BD (2 mentions)
Anti p β catenin ser 552, by Cell Signaling Technology (2 mentions)
Anti β tubulin, by Cell Signaling Technology (2 mentions)
Anti grp78, by Abcam (2 mentions)
Anti sox2, by Abcam (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Anti aldh1, by Abcam (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti phospho akt t308, by Cell Signaling Technology (1 mentions)
Anti β catenin, by BD (1 mentions)
Anti villin, by Santa Cruz Biotechnology (1 mentions)
Anti gsk 3β, by BD (1 mentions)
Anti phospho β catenin, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Anti olfm4, by Cell Signaling Technology (1 mentions)
22 protocols using anti bmi1
1
Isolation and Analysis of Mouse Colonic Epithelium
2
Immunohistochemical Analysis of OLFM4, PCNA, Lysozyme, and BMI1
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Immunohistochemistry (IHC) was performed as previously described [25 (link)]. Briefly, the 4-μm thick sections were deparaffinized in xylene, rehydrated in graded alcohol, and washed with 0.01-M PBS (pH 7.4). After the epitope retrieval with a citrate buffer (pH 6.0; Dako, Carpinteria, CA, USA) and the blocking with a blocking solution (Dako), the tissue sections were incubated with anti-OLFM4 (1:400 dilution, Cell Signalling Technology, Danvers, MA, USA), anti-PCNA (1:100 dilution, Abcam, Cambridge, UK), anti-lysozyme (1:3000 dilution, Abcam), and anti-BMI1 (1:400 dilution, Abcam) antibodies overnight at 4 °C. After washing with PBS, the sections were incubated for 30 min with horseradish peroxidase-conjugated secondary antibodies (Dako) and the antigen–antibody interaction was visualized using the chromogenic substrate 3,3′ diaminobezidine (DAB; Dako).
3
Western Blot Analysis of BMI1, P-GP, and p53
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WB was carried out base on the standard protocol, as mentioned earlier [37 (link)]. The primary antibodies were applied as followed: anti-BMI1 (Abcam, Cat. No. ab126783), anti-P-GP (Abcam, Cat. No. ab170904) and anti-p53 (Abcam, Cat. No. ab1101). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Proteintech, Cat. No. 60004-1-Ig) was used as a loading control.
4
Small Intestinal Tissue Fixation and Immunohistochemistry
5
Characterization of KSHV Latent Gene Regulation
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SLK (NIH AIDS Reagent Program) and 293T (ATCC) cells were maintained in DMEM medium supplemented with 10% FBS, and penicillin/streptomycin (P/S). BCBL-1 (NIH AIDS Reagent Program) was cultured in RPMI medium with 10% FBS and P/S. iSLK cells carrying BAC16 mutants were grown in DMEM medium with 10% FBS, P/S, 1 μg/ml puromycin, 250 μg/ml G418 and 1 mg/ml hygromycin. The origin of iSLK cell line was previously described [55 (link)]. The following antibodies were used in ChIPs and immunoblots: anti-histone H3 (Abcam ab1791), anti-H3K27me3 (Active Motif #39155), anti-H3K4me3 (Active Motif #39159), anti-EZH2 (Active Motif #39875), anti-SUZ12 (Active Motif #39357), anti-RING1B (Abcam ab3832), anti-BMI1 (Abcam ab14389), LANA (Advanced Biotechnologies #13-210-100). Anti-SPT5 and anti-CyclinT1 antibodies were purchased from Santa Cruz Biotechnology.
6
Immunoblot Analysis of Protein Markers
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Protein was extracted as described [52 (link)], quantitated using BCA assay (Pierce) and subjected to immunoblot analysis using anti-Bmi1 (Abcam; Cambridge, MA), anti-CHOP, anti-calreticulin, anti-phospho-EIF2α, anti-β-tubulin (Cell Signaling, Danvers, MA) and anti-Grp78 (Abcam). Secondary antibodies were from Santa Cruz Biotechnology (Dallas, TX). Molecular weight markers (Cat. # 10748010, 5 µL per run, or Cat. #LC5800, 10 µL per run) for immunoblot analyses were from Invitrogen (Grand Island, NY).
7
Western Blot Analysis of Stem Cell Markers
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Total proteins were extracted using RIPA lysis buffer on ice, electrophoresed on 12% SDS-PAGE gels (Bio-Rad) and blotted onto nitrocellulose membranes (Amersham Biosciences Corp, Sunnyvale, CA). The membranes were blocked with 5% non-fat milk powder at room temperature for 2 hours and incubated overnight with primary antibodies: anti-ATG4A (1:400) (Abcam, Cambridge, UK), anti-E-cadherin (1:2000) (BD Transduction Laboratories, Franklin Lakes, NJ), anti-N-cadherin (1:1000) (BD Transduction Laboratories), anti-vimentin (1:1000) (BD Transduction Laboratories), anti-Sox2 (1:500) (Abcam), anti-Oct4 (1:400) (Abcam), anti-Bmi-1 (1:400) (Abcam), anti-LC3I/II (1:1000) (Cell Signaling Technology, Danvers, MA) and anti-β-actin antibody (1: 2000) (Sigma-Aldrich). After three 5-min washes, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1: 2000) (Santa Cruz Biotechnology, Dallas, TX) for 2 hours at room temperature and then washed again in TBS-T and visualized with an enhanced chemiluminescence kit (ECL-kit) (Santa Cruz Biotechnology). All of the experiments were performed in triplicate.
8
Characterization of Breast Cancer Cell Lines
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BT474, T47D, MDA-MB-231, BT549 and SKBR3 breast cancer cell lines were purchased from the Committee on Type Culture Collection of the Chinese Academy of Science (Shanghai, China). The pEGFP-C1-ERα plasmid was purchased from Addgene (Cambridge, MA, USA), and pcDNA3-BMI1 plasmid was a gift from Dr. MH Yang (Institute of Clinical Medicine, National Yang-Ming University, Taipei city, Taiwan) [20 (link)]. A wild type BMI1 promoter (pXP2-BMI1-Luc1000) from −1023 to −1 was constructed and a mutant BMI1 promoter was generated by replacing a TGACC (−178–174) sequence in the wild type BMI1 promoter with a GACCC sequence. The following antibodies were used in this study: anti-E-cadherin (DAKO, Glostrup, Denmark; NCH-38), anti-Bmi1 (Abcam, Cambridge, UK; ab126783), anti-ERα (Santa Cruz Biotechnology, CA, USA; sc-543), anti-β-actin (Santa Cruz Biotechnology, Dallas, TX, USA; sc-4777), PE-conjugated anti-CD24 (BD Biosciences, Bedford, MA, USA; ML5), APC-conjugated anti-CD44 (BD Pharmigen, San Jose, CA, USA; G44-26), PE-conjugated mouse IgG (BD; G155-178), and APC-conjugated mouse IgG (BD; 27–35).
9
Western Blot Analysis of BMI1 Protein
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Human erythroblasts were lysed in RIPA buffer (Thermo Fisher Scientific, San Jose, CA, USA) supplemented with protease and phosphatase inhibitor cocktails (Roche, Nutley, NJ, USA), followed by centrifugation at 16,000 × g for 20 min at 4°C. Protein concentrations of cell lysates were measured using a Pierce BCA protein assay kit (Thermo Fisher Scientific). Proteins (20 μg per sample) were separated by NuPAGE 4%–12% Bis-Tris gel (Invitrogen) followed by transfer to nitrocellulose membrane (GE Healthcare) at 300 mA for 1 h. The membranes were blocked in 5% (w/v) skim milk in TBS-T (Tris-buffered saline containing 0.1% Tween 20; Bio-Rad, Hercules, CA, USA) and incubated overnight at 4°C with 1:1,000 dilution of anti-BMI1 (catalog no. ab126783, Abcam, Cambridge, MA, USA) and 1:1,000 dilution of anti-GAPDH (catalog no. 5174, Cell Signaling Technology, Danvers, MA, USA). The membrane filters were then incubated with 1:10,000 dilution of goat anti-rabbit immunoglobulin G (IgG) (catalog no. 926-32211; LI-COR Biosciences, Lincoln, NE, USA) secondary antibody for 1 h at room temperature. Detected protein bands were visualized by Odyssey Imaging Systems as we previously published.66 (link)
10
Western Blot Analysis of BMI1 Protein Expression
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Cells were lysed in RIPA buffer with protease inhibitors and phosphatase inhibitors. The protein extracts were loaded onto a 10 % sodium SDS-PAGE gel and transferred to a PVDF membrane. The blots were probed with primary antibodies (anti-BMI1, 1:1000, Abcam, Cambridge, UK) followed by the HRP-conjugated secondary antibody. Following three Tris-buffered saline containing 0.1 % Tween-20 (TBST) washes, the membranes were developed using ECL Plus (Millipore, MA, USA) and exposed to X-ray film. GAPDH served as the loading control.
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