Sc 371

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Sc-371 is a lab equipment product offered by Santa Cruz Biotechnology. It is designed to perform a core function, but a detailed description while maintaining an unbiased and factual approach is not available.

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18 protocols using sc 371

Immunoblot Analysis of Signaling Proteins

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The steady-state levels of ERK5, p-ERK5, MEK5, NF-κB, p-NF-κB, IκB-α, p-IκB-α, Vimentin, β-actin, HDAC and GAPDH were determined by immunoblot analysis, as previously described.^{44 (link)} Briefly, blots were incubated overnight at 4 °C with primary rabbit antibody reactive to ERK5 (#3372), p-ERK5 (#3371) or primary mouse antibody reactive to p-IκB-α (#9246; all from Cell Signalling) or primary rabbit antibody reactive to NF-κB (#sc-372), IκB-α (#sc-371) or primary mouse antibody reactive to MEK5 (#sc-135986), Vimentin (#sc-32322), GAPDH (#sc-32233), β-actin (#sc-8432; all from Santa Cruz Biotechnology) or HDAC (#05-614, Merck Millipore Corp, Billerica, MA, USA) or primary rabbit antibody reactive to p-NF-κB (#ab131109; Abcam). Next, immunoblots were incubated with anti-rabbit or -mouse secondary antibody conjugated with horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA, USA) for 3 h at RT. The relative intensifies of protein bands were analysed using the densitometric analysis software Image Lab version 5.1—beta, using a Chemidoc MP Imaging System for acquisition (Bio-Rad Laboratories).

Simões A.E., Pereira D.M., Gomes S.E., Brito H., Carvalho T., French A., Castro R.E., Steer C.J., Thibodeau S.N., Rodrigues C.M, & Borralho P.M. (2015). Aberrant MEK5/ERK5 signalling contributes to human colon cancer progression via NF-κB activation. Cell Death & Disease, 6(4), e1718-.

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Western Blot Analysis of Cell Signaling Proteins

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Cell extracts were prepared using RIPA lysis buffer (150 mM sodium chloride, 1% NP-40, 0.1% SDS, 50 mM Tris, pH 7.4) containing 1 mM β-glycerophosphate, 2.5 mM sodium pyrophosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate, and protease inhibitor (Roche, Basel, Switzerland). Protein concentration was quantified using Bradford assay reagent (Bio-Rad) according to manufacturer instructions. Proteins were resolved by SDS-PAGE and then transferred to a polyvinylidene fluoride membrane (Pall Corporation, Port Washington, NY, USA). Membranes were blocked with 5% non-fat milk and incubated with the following antibodies at the indicated dilutions: anti-p21 (1:500; sc-397), anti-IκBα (1:500; sc-371), anti-p53 (1:500; sc-126, all from Santa Cruz Biotechnology), anti-p-p53 (1:500; 9286, Cell Signaling Technology), anti-ID1 (1:2,000; BCH-1-195-14, Biocheck, Foster City, CA, USA), anti-ID2 (1:500, sc-489, Santa Cruz Biotechnology), anti-ID3 (1:500, sc-490, Santa Cruz Biotechnology), anti-ID4 (1:200; ab49261, Abcam), and anti-β-actin (1:10,000; A5316, Sigma-Aldrich). Membranes were then incubated with a horseradish peroxidase-conjugated anti-IgG secondary antibody (Pierce Biotechnology, Rockford, IL, USA) and visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology).

Jeon H.Y., Ham S.W., Kim J.K., Jin X., Lee S.Y., Shin Y.J., Choi C.Y., Sa J.K., Kim S.H., Chun T., Jin X., Nam D.H, & Kim H. (2019). Ly6G+ inflammatory cells enable the conversion of cancer cells to cancer stem cells in an irradiated glioblastoma model. Cell Death and Differentiation, 26(10), 2139-2156.

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Thioridazine Modulates LPS-Induced IkB-α in Macrophages

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RAW 264.7 macrophages (1 × 10⁴) were seeded on sterile coverslips. Prior to treatment, cells were allowed to synchronize by growing them in Dulbecco’s minimal essential medium (DMEM) serum-free media overnight. Cells were treated with Thioridazine (10 µM), 30 minutes prior to lipopolysaccharide (LPS) treatment (250 ng/mL) for 10 and 30 minutes respectively. After treatment, the media was aspirated and washed three times with 1X phosphate buffered saline (PBS). The cells were fixed with 4% paraformaldehyde and again washed three times with PBS (1x). Further cells were permeabilized with 0.1% Triton-X-100 for 10 minutes at room temperature. Cells were then blocked with 5% BSA in 1X PBS for 1 h at room temperature followed by incubation with primary antibody for IkB-α (sc-371, Santa Cruz Biotechnology, Inc.) for 1 h (1/200 in TBST buffer). After washing with 1X PBS, cells were stained with secondary antibody Chicken-anti rabbit-Alexa Fluor 594 (Molecular Probes-A21442) for 1 h. Nuclear counterstaining was done using DAPI (Thermo Fisher Scientific) according to manufacturer’s instructions. Stained cells were analyzed by Olympus confocal laser scanning microscope at 40X magnification and 6 times zoom.

Baig M.S., Roy A., Saqib U., Rajpoot S., Srivastava M., Naim A., Liu D., Saluja R., Faisal S.M., Pan Q., Turkowski K., Darwhekar G.N, & Savai R. (2018). Repurposing Thioridazine (TDZ) as an anti-inflammatory agent. Scientific Reports, 8, 12471.

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ABIN-1 Regulation and Signaling Assessment

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Antibodies: ABIN-1 (SC134660, Santa Cruz Biotechnology, Santa Cruz CA or 4664, Cell Signaling, Beverly MA), IκBα (SC371, Santa Cruz Biotechnology), α-tubulin (ab40742, Abcam, Cambridge MA) and β-actin (ab49900, Abcam). Secondary Abs were from Thermofisher. Inhibitors: MG132 (474790–100UG, 20μM), staurosporine (569396, 100nM), and IKK inhibitor VII (401486–1MG, 10µM EMD Millipore-Sigma, Billerica MA).
HEK293T cells were cultured in DMEM and ST2 cells and MEFs were cultured in α-MEM (Sigma, St Louis MO) containing 10% fetal bovine serum (FBS) with L-glutamine and antibiotics (Invitrogen). Transfections were performed with Fugene HD (Promega, Madison WI) or Lipojet (SignaGen, Rockville MD). Plasmids expressing ABIN-1, the ABIN-1 promoter and the Lcn2 promoter were previously described (13 (link)–15 (link)). Luciferase assays were performed as described (7 , 14 (link)). SiRNAs were from Dharmacon (GE, Lafayette CO): Tnip1 (ABIN-1) (L-047652–01), TNFαip3 (A20) (L-058907–02) and non-targeting mock (D-001810–10-20). siRNA transfections were performed 48 h prior to stimulation. In all cases efficiency of knockdown was confirmed by qPCR.

Cruz J.A., Childs E.E., Amatya N., Garg A.V., Beyaert R., Kane L.P., Aneskievich B.J., Ma A, & Gaffen S.L. (2017). Interleukin-17 signaling triggers degradation of the constitutive NF-κB inhibitor ABIN-1. ImmunoHorizons, 1(7), 133-141.

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Western Blot Analysis of HIF-1α and NF-κB

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Cells were lysed in M-PER (Thermo-Fisher Scientific) supplemented with COMPLETE protease inhibitor mixture (Roche). Total protein was quantified using the BCA Protein Assay Kit (Pierce). Equal amounts of protein were subjected to SDS-PAGE using 7.5% to 15% Tris-Glycine gradient gels, and blotted onto PVDF membranes (Bio-Rad Laboratories, Inc.). After blocking with 5% skimmed milk, membranes were incubated with primary antibodies against HIF-1α (1:1000, NB100–134, Novus Biologicals), IκBα (1:200; sc-371, Santa Cruz Biotechnology), p- IκBα^S32/36 (1:200; sc-101713, Santa Cruz Biotechnology) or Actin (1:1000, AC-74, Sigma-Aldrich) in Can Get Signal solution (Toyobo). The membranes were then incubated with HRP-conjugated secondary antibody (Promega), and immunoreactive bands were visualized with ECL plus (GE Healthcare) according to the manufacturer’s instructions. Original images of the immunoblots were shown in Supplementary Fig. S4. For quantification of bands, densitometry analysis was performed using ImageJ software (National Institutes of Health).

Okada K., Mori D., Makii Y., Nakamoto H., Murahashi Y., Yano F., Chang S.H., Taniguchi Y., Kobayashi H., Semba H., Takeda N., Piao W., Hanaoka K., Nagano T., Tanaka S, & Saito T. (2020). Hypoxia-inducible factor-1 alpha maintains mouse articular cartilage through suppression of NF-κB signaling. Scientific Reports, 10, 5425.

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Antibodies for Signaling Pathway Analysis

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The primary antibodies used were anti-rabbit antibodies against extracellular signal-regulated kinase (ERK) (9102), phosphorylated ERK (p-ERK) (9101), c-Jun N-terminal kinase (JNK) (9258), phosphorylated c-Jun N-terminal kinase (p-JNK) (9251), p38 mitogen-activated protein kinase (MAPK) (8690), phosphorylated p38 MAPK (p-p38 MAPK) (4631), Bax (2772), Bcl-2 (2870), the nuclear factor-κB (NF-κB) p65 subunit (3034), cyclin B1 (4138), phosphorylated cdc2 (Tyr 15) (9111), phosphorylated histone H3 (Ser 10) (3377), and histone H3 (9715), all of which were obtained from Cell Signaling Technology, Inc. (Beverly, MA), and anti-rabbit antibodies against nAChR α3 (NBP1-18793), nAChR α5 (NBP1-69122), and nAChR β1 (ANC-001), which were obtained from Novus Biochemicals (Littleton, CO) and Alomone Labs (Jerusalem, Israel). Anti-rabbit antibodies against IκBα (SC-371) and β-actin (A3854) were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX) and Sigma-Aldrich Co. (St. Louis, MO), respectively.

Kim C.S., Choi J.S., Joo S.Y., Bae E.H., Ma S.K., Lee J, & Kim S.W. (2016). Nicotine-Induced Apoptosis in Human Renal Proximal Tubular Epithelial Cells. PLoS ONE, 11(3), e0152591.

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Western Blot Analysis of Protein Expression

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A total of 40 µg protein extract was boiled for 10 min in SDS sample buffer, separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane by electroblotting. Non-specific reactivity was blocked in non-fat dry milk in Tween-PBS (5% (w/v) milk in phosphate-buffer saline (PBS) (pH 7.4) and 0.005% Tween 20) for 2 h at room temperature. The nitrocellulose membranes were incubated overnight at 4 °C with the following antibodies: (a) anti-FHC (H-53) (1:200; sc-25617, Santa Cruz Biotechnology, Dallas, TX, USA), (b) anti-p65 (C-20) (sc-372, 1:1000; Santa Cruz Biotechnology), (c) anti-HDAC (AV38530, 1:5000; Sigma-Aldrich), (d) anti-HA probe (F-7) (sc-7392, 1:1000; Santa Cruz Biotechnology), (e) anti-γ-Tubulin antibody (C-20) (1:3000; sc-7396, Santa Cruz Biotechnology), (f) anti-phospho-IκBα (Ser 32/36) (1:1000; sc-101713, Santa Cruz Biotechnology), and (g) anti-IκBα (C-21) (1:1000; sc371, Santa Cruz Biotechnology).
Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and immunoreactive bands were visualized with the ECL Western blotting detection system (BioRad, Hercules, CA, USA).

Aversa I., Chirillo R., Chiarella E., Zolea F., Di Sanzo M., Biamonte F., Palmieri C, & Costanzo F. (2018). Chemoresistance in H-Ferritin Silenced Cells: The Role of NF-κB. International Journal of Molecular Sciences, 19(10), 2969.

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Western Blot Analysis of NF-κB Pathway

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Aliquots from whole-cell extracts were loaded onto 7–14% SDS polyacrylamide gels (30–40 μg protein/lane) and separated by electrophoresis under reducing conditions. The proteins were transferred onto nitrocellulose membranes (Amersham, Inc., Arlington Heights, IL, USA) by electroblotting. The transfer of protein was verified using reversible staining with Ponceau S (Sigma-Aldrich, St. Louis, MO, USA). The membranes were blocked using 3% non-fat dry milk in TBS-T (Tris-buffered saline and 0.2% Tween 20) and then incubated overnight at 4 °C with antibodies for TRAF1 (sc-1831, Santa Cruz Biotechnology, Dallas, TX, USA), TRAF2 (sc-876, Santa Cruz Biotechnology, Dallas, TX, USA), IκBα (sc-371, Santa Cruz Biotechnology, Dallas, TX, USA), and actin (sc-1615, Santa Cruz Biotechnology, Dallas, TX, USA) in TBS-T solution containing 3% dry milk. After washing the membranes with TBS-T, the primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies (anti-mouse, anti-rabbit, anti-goat), and visualized by exposure to BioMax MR film (Kodak, Rochester, NY, USA) using the enhanced chemiluminescence detection system (Santa Cruz Biotechnology). Actin served as a loading control.

Park Y., Lee H., Lim J.W, & Kim H. (2019). Inhibitory Effect of β-Carotene on Helicobacter pylori-Induced TRAF Expression and Hyper-Proliferation in Gastric Epithelial Cells. Antioxidants, 8(12), 637.

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Evaluating BAF Complex Subunit Levels

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Cells were treated with BAF inhibitors for 36 h and then lysed with IP buffer (25 mM HEPES, pH 7.9, 150 mM KCl, 1 mM EDTA, 5 mM MgCl2, 5% glycerol, 1% NP40, 0.5 mM dithiothreitol and a protease inhibitor cocktail (Sigma Aldrich)) for 30 min on ice. Whole-cell protein lysate was used for SDS-PAGE in order to detect BAF250a, BRG-1, INI-1, IκB, and GAPDH. The following antibodies were used in Western blot analysis: anti-SMARCA4-BRG1 (sc-17,796, Santa Cruz Biotechnology; ab4081-100, Abcam), anti-ARID1a-BAF250a (kind gift from C.P. Verrijzer), anti-SMARCAB1-hSNF5 (ab4552, Abcam), anti- IκB (sc-371, Santa Cruz Biotechnology) and anti-GAPDH (ab8245, Abcam). Signal intensity for representative blots was quantified using ImageJ software according to NIH guidelines. Briefly, specific bands, related to protein of interest were marked in order to generate plots of relative density. Area under the plots, representing each band intensity, was normalized to its loading control and then to its untreated sample for BAF250a and INI-1, each band intensity for BRG-1 was normalized to its corresponding loading control and to untreated control, band intensity of treated samples for GAPDH was normalized with untreated controls.

Stoszko M., De Crignis E., Rokx C., Khalid M.M., Lungu C., Palstra R.J., Kan T.W., Boucher C., Verbon A., Dykhuizen E.C, & Mahmoudi T. (2015). Small Molecule Inhibitors of BAF; A Promising Family of Compounds in HIV-1 Latency Reversal. EBioMedicine, 3, 108-121.

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MBP Isomers Regulate Protein Expression

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Following incubation in the presence or absence of MBP charge isomers (C1 or C8, 0.5 M), cells were subjected to Western blotting according to the method described in the initial study.²² The following primary antibodies were used: anti-EAAT-2 (ab41621, Abcam), anti-HMGB1 (ab18256, Abcam), anti-PPAR-γ (ab191407, Abcam), anti-ikB (sc-371, Santa Cruz Biotechnology, USA). Immunoreactivity was detected by enhanced chemiluminescence autoradiography (ECL kit, Santa-Cruz Biotechnology). Protein concentrations were determined using a BCA protein assay kit (Thermo Scientific). Signal quantification from Western blots was assisted by ImageJ software.

Chikviladze M., Mamulashvili N., Sepashvili M., Narmania N., Ramsden J., Shanshiashvili L, & Mikeladze D. (2023). Citrullinated isomer of myelin basic protein can induce inflammatory responses in astrocytes. IBRO Neuroscience Reports, 16, 127-134.

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