Trichostatin A (TSA, T1952), Drabkin's reagent (D5941), and an antibody against Flag (F1804) were purchased from Sigma-Aldrich (St. Louis, MO). Human vascular endothelial growth factor (VEGF165, 100–20) was purchased from PeproTech Inc. (Rocky Hill, NJ). Dihydroethidium (DHE, D1168) was obtained from Invitrogen (Carlsbad, CA). Antibodies against Bach1 (Santa Cruz #sc-271211, RRID:AB_10608972), NQO1 antibody (Santa Cruz #sc-32793, RRID:AB_ 628036), normal mouse IgG (sc-2025, RRID:AB_737182), normal rabbit IgG (sc-66931, RRID:AB_1125055), normal goat IgG (sc-2028, RRID:AB_737167), β-actin (sc-47778, RRID:AB_626632), HA (sc-7392, RRID:AB_627809) and Protein A/G PLUS-Agarose (sc-2003, RRID:AB_10201400) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against CD31 (ab28364, RRID:AB_726362) was purchased from Abcam (Cambridge, MA). GST antibody (#66001, RRID:AB_10951482), GFP-tag antibody (Proteintech #50430–2-AP, RRID:AB_11042881) and HO-1 antibody (Proteintech #10701–1-AP, RRID:AB_2118685) was purchased from Proteintech (Rosemont, IL).
Ho 1 antibody
Manufactured by Proteintech
Sourced in United States, China
The HO-1 antibody is a laboratory-grade reagent used to detect and quantify the expression of the heme oxygenase-1 (HO-1) protein in various biological samples. HO-1 is an enzyme involved in the degradation of heme, a critical component of hemoglobin. This antibody can be used in techniques such as Western blotting, immunohistochemistry, and flow cytometry to study the role of HO-1 in cellular processes and disease states.
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Lab products found in correlation
Nrf2 antibody, by Proteintech (4 mentions)
Gapdh antibody, by Proteintech (3 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Keap1 antibody, by Proteintech (2 mentions)
Nqo 1 antibody, by Abcam (2 mentions)
Normal goat igg, by Santa Cruz Biotechnology (1 mentions)
Nqo1 antibody, by Santa Cruz Biotechnology (1 mentions)
Gst antibody, by Proteintech (1 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
γh2ax antibody, by Abcam (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh antibody, by Bioworld Technology (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
β actin antibody, by Abcam (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Nrf2 phospho s40, by Abcam (1 mentions)
Nqo1 antibody, by Proteintech (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Tlr4 antibody, by Proteintech (1 mentions)
Ab 8 macroporous resin, by Yuanye Bio-Technology (1 mentions)
0.22 m nitrocellulose membrane, by GE Healthcare (1 mentions)
8 protocols using ho 1 antibody
1
Molecular Mechanisms of Endothelial Regulation
2
Western Blot Analysis of DNA Damage and Oxidative Stress Markers
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The cells were lysed with radio immunoprecipitation assay (RIPA) lysate buffer (Cell Signal Technology, Danvers, MA, USA) to obtain the total protein. After centrifugation at 13,000× g for 10 min, the protein concentration was measured using the bicinchoninic acid (BCA) protein quantification kit. Protein was separated on a 10% or 12% SDS-PAGE gel and then blotted onto a PVDF membrane. After blocking with 5% BSA for 2 h, the membrane was incubated overnight at 4 °C with the following primary antibodies: γ-H2AX antibody (abcam, Cambridge, England), 8-oxoguanine DNA glycosylase (OGG1) antibody (Proteintech, Wuhan, China), poly (ADP-ribose) polymerase-1 (PARP1) antibody (Proteintech, Wuhan, China), X-ray repair cross-complementing gene 1 (XRCC1) antibody (Proteintech, Wuhan, China), HO-1 antibody (Proteintech, Wuhan, China), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Bioworld, Nanjing, China), and β-actin antibody (abcam, Cambridge, England). The secondary antibody was RDye800® conjugated goat anti-rabbit IgG (KPL, Gaithersburg, MD, USA) and RDye800® conjugated goat anti-mouse IgG (KPL, Gaithersburg, MD, USA), incubated at room temperature for 1 h. Finally, the bands were obtained using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA).
3
Oxidative Stress Signaling Pathway
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RPMI 1640 and fetal bovine serum were purchased from Gibco-BRL (Gibco-BRL, Carlsbad, California, USA). Active oxygen detection kit (S0033), total SOD active detection kit (S0101), and lipid oxidation detection kit (S0131) were purchased from Full-Style Gold (Beijing, China). The enhanced BCA protein kit (P0009) and nuclear cytoplasmic protein extraction kit (P0028) were purchased from Beyotime Biotechnology (Shanghai, China). Keap1 antibody (catalog number 60027-1-lg), Nrf2 antibody (catalog number 66504-1-lg), NQO1 antibody (catalog number 67240-1-lg), and HO-1 antibody (catalog number 67643-1-lg) are available for purchase from Proteintech (USA). Nrf2 (phospho S40) (catalog number EP1809Y) was purchased from Abcam (UK). β-Actin (Cat. No. 12620), anti-rabbit IgG HRP conjugate (V7951), and anti-mouse IgG HRP conjugate (W4021) were purchased from Promega Corporation (Madison, W1, USA). The inhibitor ML385 (catalog number HY-100523) was purchased from MCE (Shanghai, China). All other reagents for this study were of analytical grade.
4
Hepatoprotective Effects of HTF
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The HTF was obtained by the method of Cheng et al. with some modifications. 95% ethanol (analytical-grade) was purchased from the Chengdu Chron Chemicals Co., Ltd. (Chengdu, Sichuan, China). The AB-8 macroporous resin was purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Cytokines in serum and liver (interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α)) were measured using commercial kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Both serum and liver biochemical indicators (high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), triglyceride (TG), total cholesterol (TC), glutamic oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT)) were measured using Nanjing Jiancheng commercial kits. Antibodies in the experiment were purchased from Proteintech Group, Inc. (Wuhan, Hubei, China), including the Nrf2 antibody, HO-1 antibody, TLR4 antibody, MyD88 antibody, GAPDH antibody, and Keap1 antibody.
5
Analysis of A1M and HO-1 Protein Expression
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To analyze A1M secretion into the cell culture medium, equal volumes of cell culture supernatants were electrophoresed on 12% Tris-glycine SDS-PAGE gels, then proteins were transferred to 0.22 µm nitrocellulose membrane (GE Healthcare, Chicago, IL, USA) and blocked with 5% w/v milk for 60 min; they were then probed for A1M antibody (Abcam, Cambridge, UK; dilution 1:2500) at 4 °C overnight, followed by incubation with horse-radish peroxidase (HRP)-conjugated goat anti-rabbit antibody (Cell Signaling Technologies, Danvers, MA, USA) at room temperature for 1 h. The antigen–antibody complex was detected by a WesternBright ECL HRP substrate (Advansta, Menlo Park, CA, USA). To ascertain equivalent protein loading in the samples, membranes were stripped and reprobed again with a mouse anti-bovine serum albumin antibody (Abcam, Cambridge, UK). To analyze HO-1 expression, cells were lysed with a radioimmunoprecipitation assay buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 1% Sodium-deoxycholate, 0.1% SDS, 1 × Complete Mini Protease Inhibitor Cocktail) and analyzed by immunblot, as described above, using HO-1 antibody (Proteintech Group, Manchester M3 3WF, UK; dilution 1:8000), then reprobed with a mouse GAPDH antibody (Proteintech Group, Manchester, UK; dilution 1:10,000).
6
Protein Phosphorylation Profiling in NP Cells
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Human NP cells were harvested using iced-cold lysis buffer (Cell Signaling Technology, Danvers, MA, USA). For detection of phosphorylation proteins level, the total protein was extracted at 15 min after treatment, while the other treatment duration was 24 h. Protein concentration was determined using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Immunolabeling was detected using the Pierce ECL western blotting substrate (Pierce Biotechnology, Rockford, IL, USA). The following antibodies and dilutions were used: HO-1 antibody (1:500) (Proteintech), β-actin (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); phospho-ERK1/2 (1:1000), ERK1/2 (1:1000), phospho-p38 (Thr180/Tyr182) (1:1000), p38 (1:1000), phospho-JNK (T183/Y185) (1:1000), JNK (1:1000), phospho-MEK1/2 (1:1000), MEK1/2 (1:1000) and Ras (1:1000) (Cell Signaling Technology).
7
Ferroptosis-related Protein Regulation
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Ferric ammonium citrate (USA, 1185-57-5) was purchased from Sigma–Aldrich. Recombinant mouse IL-1β (Minneapolis, USA, # 401-ML) was purchased from R&D systems. Erastin (USA, S7242) and Ferrostatin-1 (USA, S7243) were purchased from Selleck. Reactive Oxygen Species Assay Kit (China, S0033) was purchased from Beyotime. C11 BODIPY Lipid Peroxidation Sensor (USA, D3861) was purchased from ThermoFisher. GPX4 (UK, ab125066, diluted 1:5000) antibody, P53 antibody (UK, ab131442, diluted 1:1000), SLC7A11 antibody (UK, ab175186, diluted 1:5000), ACSL4(UK, ab155282, diluted 1:10,000) antibody, Collagen II (UK, ab34712, diluted 1:200 for immunohistochemical staining) antibody, Collagen II (UK, ab185430, diluted 1:1000 for western blot) antibody, NQO-1 antibody (UK, ab80588, diluted 1:10,000), and Trx (UK, ab133524, diluted 1:10,000) antibody were purchased from Abcam. Nrf2 antibody (USA, 16396-1-AP, diluted 1:1000), HO-1 antibody (USA, 10701-1-AP, diluted 1:1000), Beta-actin antibody (USA, 66009-1-Ig, diluted 1:1000) and GAPDH antibody (USA, 60004-1-Ig, diluted 1:1000) were purchased from Proteintech.
8
Therapeutic Effects of MPS and VO-OHpic
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MPS was purchased from Pfizer and dissolved in sterile normal saline. VO-OHpic was purchased from selleck (USA, S8174) and dissolved in DMSO. And the final concentration of DMSO is the same (0.1%) in each group including the control. CD31 antibody (UK, ab182981), CD34 antibody (UK, ab81289), VEGF Receptor 2 (VEGFR2) antibody (UK, ab11939), VEGF Receptor 1 (VEGFR1) antibody (UK, ab32152), CD133 antibody (UK, ab19898), SIRT-1 antibody (UK, ab189494), NQO-1 antibody (UK, ab80588), and Trx antibody (UK, ab133524) were purchased from Abcam. FITC-conjugated mouse anti-rabbit IgG antibody was purchased from Boster (China, BM2012). Bad antibody (USA, #9268, for IP), Phospho-Bad antibody (USA, #5284), Bcl-xL antibody (USA, #2764), AKT antibody (USA, #4691), p-AKT antibody (USA, #4060), cleaved caspase-3 (Asp175) antibody (USA, #9661), cleaved caspase-9 antibody (USA, #9508), MFF antibody (USA, #84580), and normal rabbit IgG (USA, #2729) were purchased from Cell Signaling Technology. Bax antibody (USA, 50599-2-Ig, for western blot), Cytochrome C antibody (USA, 66264-1-Ig), Nrf2 antibody (USA, 16396-1-AP), Bcl-xL antibody (USA, 26967-1-AP), Bad antibody (USA, 10435-1-AP), FIS1 antibody (USA, 10956-1-AP), VEGF antibody (USA, 19003-1-AP), HO-1 antibody (USA, 10701-1-AP), and GAPDH antibody (USA, 60004-1-Ig) were purchased from Proteintech.
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