Total protein was extracted with NE-PER nuclear and cytoplasmic extraction reagents (Thermo). Nuclear proteins were isolated with a mammalian nuclear and cytoplasmic protein extraction kit (TransGen Biotech). Next, 50–100 μg aliquots of protein were separated on 10% polyacrylamide-SDS gels (Pplygen) and transferred to Immobilon™-P membranes (Millipore). After blocking with TBS/5% nonfat dry milk (Pplygen) for 1 h, the membrane was incubated with antibodies against mouse PPARγ (Cell Signaling Technology), histone H3 (Bioworld), and β-actin (Sigma) overnight at 4 °C. Next, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Pierce, Malibu, CA, USA) for 1 h at room temperature. Antibody binding was visualized with an enhanced chemiluminescence kit, according to the manufacturer’s protocols (Pierce).
Histone h3
Manufactured by Bioworld Technology
Sourced in United States
Histone H3 is a core histone protein that plays a fundamental role in the organization and structure of chromatin in eukaryotic cells. It is responsible for the packaging of DNA into nucleosomes, the basic unit of chromatin.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Bioworld Technology (4 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Phospho ikkα β ser176 180, by Cell Signaling Technology (2 mentions)
Nf κb p65, by Cell Signaling Technology (2 mentions)
Gapdh, by Bioworld Technology (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence solution, by Thermo Fisher Scientific (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Sirt1, by Santa Cruz Biotechnology (2 mentions)
Un scan it 6, by Silk Scientific (2 mentions)
Mammalian nuclear and cytoplasmic protein extraction kit, by Transgene (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Recombinant human egf, by Thermo Fisher Scientific (1 mentions)
Mk 2206, by Selleck Chemicals (1 mentions)
Gapdh, by Proteintech (1 mentions)
Ab76302, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
10 protocols using histone h3
1
Western Blot Analysis of PPARγ
2
Signaling Pathways in Epithelial-Mesenchymal Transition
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The primary antibodies and their commercial suppliers are YAP1 (cat. 14074S), p‐YAP1 (s127) (cat. 13008S), E‐cadherin (cat. 3195S), Snail (cat. 3895S), AKT (cat. 4691S), p‐ATK (s473) (cat. 4060S), EGFR (cat. 2646S) and p‐EGFR (Tyr1068) (cat. 3777S) from Cell Signaling Technolog, Vimentin (cat. 10366–1‐AP) from Proteintech (USA), GAPDH (cat. MB001‐100) from Bioworld, Histone H3 (cat. AF0009) from Beyotime (CHN), Goat anti‐Rabbit IgG‐H&L (Alexa Fluor® 488) (cat. ab150077) from Abcam. Recombinant human EGF was purchased from Peprotech (cat. AF‐100–15–100, Peprotech). MK2206 was purchased from Selleck (cat. S1078, Selleck) and DAPI (D1306) from Thermo Fisher.
3
Protein Analysis of Breast Cancer Cells
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Briefly, breast cancer cells, after transfection or without processing, were harvested and lysed by using a RIPA lysis buffer (Beyotime). Protein concentration was determined with the BCA protein assay kit (Beyotime). Then, the protein was separated on SDS-PAGE gels and transferred to PVDF membranes (Millipore). Membranes were blocked by 5% nonfat milk, then incubated with primary antibodies overnight at 4 °C and with corresponding secondary antibodies afterward. Detailed information about the antibodies used in this study was shown as follows: KIFC2 (PA5-40813; Invitrogen), NF-κB p65 (ab16502; Abcam), p-p65 (phospho S536) (ab76302), glyceraldehyde-3-phosphate dehydrogenase (ab8226; Abcam), Histone H3 (BS1174; Bioworld). Protein bands were visualized with ECL.
4
Protein Extraction and Western Blot Analysis
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Total proteins were extracted using the mix of RIPA lysis buffer (Beyotime, China) and PMSF (Solarbio, China). Western blot was performed with the specific antibody, C5aR1 (1:2000, Proteintech), β-actin (1:1000, Beyotime), E-cadherin (1:2000, Cell Signaling Technology), Vimentin (1:2000, Bioworld), β-catenin (1:5000, Abcam), Histone H3 (1:2000, Bioworld).
5
Western Blot Analysis of Protein Targets
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HPMECs were harvested and lysed with lysis buffer for 30 min on ice. After centrifugation for 30 min at 4 °C, the supernatants were collected and protein concentration was determined by a bicinchoninic acid (BCA) protein assay kit (Pierce, USA). Equal amounts of protein were separated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, electrotransferred to polyvinylidene di uoride (PVDF) membranes. Membrane was blocked with 5% BSA and incubated with the primary antibodies, followed by HRP-conjugated goat anti-rabbit secondary antibodies. Prohibitin and mtTFA, Phospho-IKKα/ β(Ser176/180), IκB-α, NF-κB p65, IκB-α (Cell Signaling Technology), histone H3 (bioworld) and GAPDH (bioworld) antibody was used in this study.
6
Quantitative Protein Expression Analysis
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HPMECs were harvested and lysed with lysis buffer for 30 min on ice. After centrifugation for 30 min at 4°C, the supernatants were collected and protein concentration was determined by a bicinchoninic acid (BCA) protein assay kit (Pierce, USA). Equal amounts of protein were separated by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis, electrotransferred to polyvinylidene difluoride (PVDF) membranes. Membrane was blocked with 5% BSA and incubated with the primary antibodies, followed by HRP-conjugated goat anti-rabbit secondary antibodies. Prohibitin and mtTFA, Phospho-IKKα/β (Ser176/180), IκB-α, NF-κB p65, IκB-α (Cell Signaling Technology), histone H3 (bioworld) and GAPDH (bioworld) antibody was used in this study.
7
Protein Extraction and Western Blot Analysis
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Whole cell protein extraction, cytosolic protein extraction, and nuclear protein extraction were extracted according to previous studies (Pan et al., 2014 (link); Zhang et al., 2016a (link)). Equal protein amounts were separated by polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked in 5% skim milk for 2 h at room temperature and then incubated with primary antibodies against caspase-3 (1:400, cat #9661, Cell Signaling), SIRT1 (1:200, cat #SC-15404, Santa Cruz), HMGB1 (1:1000, cat #3935S, Cell Signaling), TLR4 (1:200, cat #sc-30002, Santa Cruz), Myd88 (1:200, cat #sc-11356, Santa Cruz), Nf-κB p65 (1:200, cat #sc-372, Santa Cruz), β-actin (1:3000, cat #AP0060, Bioworld Technology, Minneapolis, MN, USA), and Histone H3 (1:3000, cat #BS7416, Bioworld Technology) overnight at 4°C. The membrane was incubated with horseradish peroxidase (HRP)-conjugated IgG for 2 h at room temperature. Detection was performed by enhanced chemiluminescence solution (Thermo Fisher Scientific, Waltham, MA, USA). Band density was quantified with UN-Scan-It 6.1 software (Silk Scientific Inc., Orem, UT, USA).
8
Western Blot Analysis of Inflammatory Markers
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The brain samples and the primary microglia in transwell upper chambers and neurons in the plates were collected for Western blotting. In brief, the extracted proteins were separated by Tris-glycine SDSPAGE and then transferred to PVDF membranes for 30 min. Primary antibodies used were Nrf2 (1 : 1000, cat# ab31163, Abcam), HO-1 (1 : 1000, cat# ab13243, Abcam), NLRP3 (1 : 200, cat# SC-66846, Santa Cruz Biotechnology), adaptor apoptosis-related speck-like protein (ASC) (1 : 200, cat# SC-22514, Santa Cruz Biotechnology), caspase-1 (1 : 200, cat# SC-56036, Santa Cruz Biotechnology), caspase-1 p20 (1 : 200, cat# SC-398715, Santa Cruz Biotechnology), Histone H3 (1 : 3000, cat# BS7416, Bioworld Technology), and β-actin (1 : 3000, cat# AP0060, Bioworld Technology, Minneapolis, MN, USA). Then, the membranes were incubated for 2 h at room temperature with corresponding second antibody. Detection was conducted by using chemiluminescence solution.
9
Protein Extraction and Western Blot Analysis
10
Lipid Metabolism Regulatory Pathway Exploration
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Pioglitazone (PIO) was purchased from MSD Pharma (Singapore) Pte. Ltd. Ezetimibe (EZE) was purchased from DEYUAN PHARM Co. Ltd. (Shanghai, China). The antibodies of SREBP-2(sc-13,552), CYP7A1 (sc-293,193), FXR (sc-25,309), LXR (sc-271,064), BSEP (sc-74,500), CCK-AR(sc-514,303) and NPC1L1(sc-166,802) were purchased from Santa Cruz Biotechnology Co. Ltd. (Santa Cruz, CA). The antibodies of HMGCR (ab174830), ABCG5 (ab87116) and ABCG8 (ab126493) were from Abcam Technology Co. Ltd. (Cambridge, UK). The antibodies of ACAT2 (13294S) and PPARγ (2443S) were from Cell Signaling Technology Inc. (Boston, USA). Histone H3, β-actin and the secondary antibodies were from Bioworld Technology Co. Ltd. (Minneapolis, MN, USA). BCA assay kit was from Beyotime (Nanjing China). Amplex Red cholesterol (CH) assay kit was from the Invitrogen (USA). All other chemical or Biological reagents were commercially available.
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