Sc 10789

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Sc-10789 is a laboratory equipment product offered by Santa Cruz Biotechnology. It is designed for use in scientific research applications. The core function of this product is to facilitate [core function]. No further details or interpretations about the intended use of this product can be provided in an unbiased and factual manner.

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Close spelling variants of this product

Anti-HO-1 (sc-10789 HO-1 (sc-10789)

8 protocols using sc 10789

Evaluating Antioxidant Enzyme Expression

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To evaluate HO-1, NQO1 and SOD expression, IEC-6 cells (2 × 10⁴ cells/well; 96-well plates) were treated with IS (31.2–250 μM) for 24 h.
Thereafter, the cells were collected, washed with PBS, and incubated with Fixing Solution for 20 min at 4 °C and successively for 30 min at 4 °C with Fix Perm Solution. Anti-HO-1 (sc-10789, Santa Cruz Biotechnology) or anti-NQO1 (sc-376023, Santa Cruz Biotechnology) or anti-SOD (sc-30080, Santa Cruz Biotechnology) antibody were subsequently added. The secondary antibody was added, for 30 min, in Fix Solution, and cell fluorescence was evaluated by FACSscan (Becton Dickinson) and elaborated with Cell Quest software, as previously reported [60 (link)].

Adesso S., Ruocco M., Rapa S.F., Dal Piaz F., Di Iorio B.R., Popolo A., Autore G., Nishijima F., Pinto A, & Marzocco S. (2019). Effect of Indoxyl Sulfate on the Repair and Intactness of Intestinal Epithelial Cells: Role of Reactive Oxygen Species’ Release. International Journal of Molecular Sciences, 20(9), 2280.

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Western Blotting of Cellular Proteins

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PC12 cells were incubated in 6-well plates at a density of 3×10⁵ and incubated for 24–48 h at 37 °C. After suitable treatments, the cells were collected and lysed with ice-cold RIPA lysis buffer. Equal amounts of protein samples (80 μg) were separated on a 10% SDS-PAGE gel and transferred electrophoretically onto nitrocellulose membrane. Subsequently, the membranes were blocked with 5% skim milk and incubated overnight at 4 °C with primary antibody: anti-HO-1 (sc-10789, Santa Cruz Biotechnology, 1:300), anti-GCLC (ab-80841, abcam Biotechnology, 1:600), anti-GADPH (sc-47724, Santa Cruz Biotechnology, 1:1000), anti-β-actin (AP0060, Bioworld Technology, 1:3000). After being washed three times with 1× TBST, the membrane was incubated with anti-rabbit IgG for 1 h at room temperature. Proteins were visualized by exposure in a ChemiDoc XRS+system (Bio-Rad, Hercules, CA, USA). Band density was quantified by Image J software (National Institute of Health, Bethesda, MD, USA).

Wang J., Huang L., Cheng C., Li G., Xie J., Shen M., Chen Q., Li W., He W., Qiu P, & Wu J. (2019). Design, synthesis and biological evaluation of chalcone analogues with novel dual antioxidant mechanisms as potential anti-ischemic stroke agents. Acta Pharmaceutica Sinica. B, 9(2), 335-350.

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Synthesis and Characterization of RRx-001

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RRx-001 was obtained from ATK Aerospace Systems [10 ]. The synthesis and characterization of RRx-001 is reported in detail elsewhere [8 –12 (link)]. For in vitro cell culture experiments, RRx-001 was dissolved in DMSO and then diluted with growth medium with a final concentration of DMSO at < 0.05%. For animal experiments, RRx-001 formulation was prepared by dissolving 10 mg RRx-001 in 0.5 mL DMA-PEG 400 (1:2) and then diluting with double distilled water to obtain a 2 mg/mL solution for injection.
The pcDNA-ARE-FLUC and pcDNA-CMV-RLUC-mRFP vectors were constructed in our lab. Nrf2-specific small interfering RNA (Nrf2-siRNA, sc-37049), scrambled siRNA (sc-37007), transfection reagents (sc-29528 and sc-36868), and antibodies against Nrf2 (sc-13032), HO-1 (sc-10789), NQO1 (sc-393736), Δ-actin (sc-130656) and lamin-B (sc-365962) were purchased from Santa Cruz Biotechnology, Santa Cruz, CA.

Ning S., Sekar T.V., Scicinski J., Oronsky B., Peehl D.M., Knox S.J, & Paulmurugan R. (2015). Nrf2 activity as a potential biomarker for the pan-epigenetic anticancer agent, RRx-001. Oncotarget, 6(25), 21547-21556.

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Western Blotting of Oxidative Stress Markers

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Western blotting was performed following standard procedures. Anti-Nrf2 antibody (1 : 100, AP52269, Abgent), anti-Brg1 antibody (1 : 100; ab110641, Abcam), anti-NQO1 antibody (1 : 100; ab28947, Abcam), anti-HO-1 antibody (1 : 100; sc-10,789, Santa Cruz), and secondary antibody (1 : 2000; Millipore) were used to detect protein expression. Anti-β-actin and anti-lamin B2 (Proteintech) were used at 1 : 5000. Images were acquired by a Tanon 5500 imaging system (Tanon, Shanghai). The images were scanned with the ImageJ scanning software, and the data were expressed as relative values to sham or control values.

Ge M., Chen C., Yao W., Zhou S., Huang F., Cai J, & Hei Z. (2017). Overexpression of Brg1 Alleviates Hepatic Ischemia/Reperfusion-Induced Acute Lung Injury through Antioxidative Stress Effects. Oxidative Medicine and Cellular Longevity, 2017, 8787392.

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Estrogen Signaling Regulation in MCF-7 Cells

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MCF-7 cells were seeded in 100 mm dishes at a density of 500,000 cells per dish and treated with 0.1% DMSO or 100 pM E2 in the presence or absence of 10 μM LH1092, 10 μM LH1095, or 100 nM CDDO-IM. After 24 hours of treatment, cells were lysed in RIPA buffer containing 1% protease and phosphatase inhibitor. Cell lysates were separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membranes were incubated with primary antibodies at 4°C overnight and with secondary antibodies at room temperature for 1 hour. Primary antibodies against PGR (1:500, 8757), c-MYC (1:1000, 5605), TFF1/pS2 (1:1000, 15571), and Cathepsin D (1:1000, 2284) were from Cell Signaling Technology (Danvers, MA). Primary antibodies against HO-1 (1:200, sc-10789) and NQO1 (1:100, sc-271116) were from Santa Cruz Biotechnology (Dallas, TX). β-actin antibody (1:2000, A1978) was from Sigma Aldrich (Saint Louis, MO). Anti-rabbit and anti-mouse secondary antibodies were from Cell Signaling Technology (Danvers, MA). β-actin was used as a loading control.

Xie T., Zahid H., Ali A.R., Joyce R., Yang G., Winz C., Le Y., Zhou R., Furmanski P., Hu L, & Suh N. (2023). Inhibitors of Keap1-Nrf2 Protein-Protein Interaction Reduce Estrogen Responsive Gene Expression and Oxidative Stress in Estrogen Receptor-Positive Breast Cancer. Toxicology and applied pharmacology, 460, 116375.

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Immunofluorescence Imaging of HRMECs

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HRMECs were cultured in coverslips in six-well plates and starved in minimal media overnight. They were left untreated or were treated with 100 ng/ml of cytokine-HMGB1, lipopolysaccharide free (Cat. No. REHM120, IBL International Corporation) for 24 h and were fixed with ice-cold acetone or 4% paraformaldehyde followed by blocking with 10% goat serum for 1 h. Without being washed, the cells were incubated with anti-HO-1 (1:100, sc-10789, Santa Cruz Biotechnology, Inc.), anti-VAP-1 (1:100, ab196739, Abcam), or anti-8OHdG (1:50, ab62623, Abcam) primary antibodies overnight at 4 °C. After washing, cells were incubated with secondary antibodies conjugated with appropriate fluorophores (Alexa Fluor 488 or 555) diluted in 10% goat serum (1:1000) for 1 h at room temperature. After washing, the cell-containing coverslips were counterstained with DAPI before being mounted on a glass slide with the use of the UltraCruz^TM Mounting Medium (sc-24941, Santa Cruz Biotechnology Inc.). Immunofluorescence microscopy was performed using an Olympus motorized system microscope (BX61, Olympus Corporation, Tokyo, Japan) at 20X or 40X magnification.

Abu El-Asrar A.M., Alam K., Garcia-Ramirez M., Ahmad A., Siddiquei M.M., Mohammad G., Mousa A., De Hertogh G., Opdenakker G, & Simó R. (2017). Association of HMGB1 with oxidative stress markers and regulators in PDR. Molecular Vision, 23, 853-871.

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Protein Analysis by SDS-PAGE and Western Blot

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Protein analysis was performed by SDS-PAGE electrophoresis/western blot, loading 30 μg each sample. Proteins were transferred to a nitrocellulose membrane (Amersham, GE Healthcare Europe GmbH, Milano, Italy), and revealed by immunoblotting with specific antibodies: rabbit polyclonal heme oxygenase-1 (HO-1) (1:200)(sc-10789; Santa Cruz Biotechnology™, Dallas, TX, USA), rabbit polyclonal inducible nitric oxide synthase (iNOS) (1:200) (sc-8310; Santa Cruz Biotechnology™), rabbit polyclonal cytochrome 1b1 (Cyp1b1) (1:200) (sc-32882; Santa Cruz Biotechnology™), goat polyclonal heat shock protein 70 (Hsp70) (1:200)(sc-10-70; Santa Cruz Biotechnology™), rabbit polyclonal cyclooxygenase 2 (COX2) (1:1000)(#4842; Cell Signalling Technology^®, Danvers, MA, USA), and rabbit polyclonal myeloperoxidase (MPO) (1:200) (sc-16128-R; Santa Cruz Biotechnology™). The secondary antibodies were appropriate horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:5000) (31460 Thermofisher Scientific™ Waltham, MA, USA) or donkey anti-goat (1:2000) (sc-2020; Santa Cruz Biotechnology™). Immunoblot bands have been analyzed and the optical density quantified by LAS4000 (GE Healthcare, Marlborough, MA, USA); all the data have been normalized to Ponceau staining (Sigma Chemical Co., Milano, Italy) [78 (link)].

Farina F., Lonati E., Milani C., Massimino L., Ballarini E., Donzelli E., Crippa L., Marmiroli P., Botto L., Corsetto P.A., Sancini G., Bulbarelli A, & Palestini P. (2019). In Vivo Comparative Study on Acute and Sub-acute Biological Effects Induced by Ultrafine Particles of Different Anthropogenic Sources in BALB/c Mice. International Journal of Molecular Sciences, 20(11), 2805.

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Western Blot Protocol for Protein Detection

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Whole cell lysates were derived from cells or tissues using radioimmunoprecipitation assay (RIPA) lysis buffer. Proteins (20–40 μg) with SDS-loading buffer were loaded onto gels and run on SDS-PAGE; then, proteins were transferred to the polyvinylidene fluoride (PVDF) membrane. The PVDF membranes were incubated with primary antibodies against Pro-caspase-1 (1:2000, #ab179515, Abcam), caspase-1 (1:2000, #ab108362, Abcam), NLRP3 (1:2000, #ab16097, Abcam), IL-1β (1:1000, #AF-401-NA, R&D), HO-1 (1:500, #sc-10789, Santa Cruz), GAPDH (1:500, #sc-47724, Santa Cruz), β-actin (1:500, #sc-81178, Santa Cruz), Tubulin (1:1000, #556321, BD Pharmingen), FXRa (1:2000, #ab187735, Abcam), Cyp8b1 (1:2000, #ab175843, Abcam), Cyp27a1 (1:1000,#ab126785, Abcam), Sult2a1 (1:500, #sc-376629, Santa Cruz), Bsep (1:2000, #ab155421, Abcam), Mrp3 (1:500, #sc-5776, Santa Cruz), Ntcp (1:2000, #ab131084, Abcam), and BAAT (1:2000, #ab83882, Abcam). Then, appropriate horseradish peroxidase (HRP)-linked secondary antibodies were incubated for further 1 hour at room temperature. Luminal and H₂O₂ substrates were used for chemiluminescence detection.

Chen W., Ding M., Ji L., Yao J., Guo Y., Yan W., Yu S., Shen Q., Huang M., Zheng Y., Lin Y., Wang Y., Liu Z., Lu L, & Jin X. (2023). Bile acids promote the development of HCC by activating inflammasome. Hepatology Communications, 7(9), e0217.

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