Anti bcl 3

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Anti-Bcl-3 is a laboratory reagent used for research purposes. It is an antibody that specifically binds to the Bcl-3 protein, which is a member of the NF-κB inhibitor family. The core function of Anti-Bcl-3 is to facilitate the detection and study of Bcl-3 in various experimental systems.

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Lab products found in correlation

Anti p50, by Santa Cruz Biotechnology (3 mentions) Alexa fluor 680, by Thermo Fisher Scientific (2 mentions) Anti gapdh, by Santa Cruz Biotechnology (2 mentions) Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions) Anti actin, by Merck Group (2 mentions) Ubiquitin, by Santa Cruz Biotechnology (1 mentions) Vimentin, by Santa Cruz Biotechnology (1 mentions) N cadherin, by Santa Cruz Biotechnology (1 mentions) Phos smad2, by Cell Signaling Technology (1 mentions) Anti k63 ubiquitin, by Merck Group (1 mentions) Donkey anti goat, by Thermo Fisher Scientific (1 mentions) Flag tag (flag), by Santa Cruz Biotechnology (1 mentions) K48 ubiquitin, by Merck Group (1 mentions) Smad2, by Cell Signaling Technology (1 mentions) Smad3, by Cell Signaling Technology (1 mentions) Lamin a c, by Cell Signaling Technology (1 mentions) C myc, by Santa Cruz Biotechnology (1 mentions) Alexa fluor 488, by Thermo Fisher Scientific (1 mentions) E cadherin, by Santa Cruz Biotechnology (1 mentions) Cyclin d1, by Santa Cruz Biotechnology (1 mentions)

9 protocols using anti bcl 3

Immunohistochemical and Immunofluorescence Analysis of Bcl3 in AKI

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Immunohistochemistry was carried out in 3-μm-thick paraffin-embedded tissue sections as previously described.^{37 (link)} The samples were incubated with primary anti-Bcl3 (1:100 Santa Cruz Biotechnology) antibody overnight at 4 °C. Secondary horseradish peroxidase-conjugated antibody was applied on the samples for 1 h. The sections were counterstained with Carazzi’s hematoxylin. The samples were dehydrated and mounted in Depex. Images were analyzed with Image-Pro Plus software (Media Cybernetics, Silver Spring, MD, USA).
For immunofluorescence, the samples were incubated with anti-Bcl3 (1:100 Santa Cruz Biotechnology) overnight at 4 °C, followed by a secondary Alexa Fluor 488- or 633-conjugated antibodies for 1 h. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole. After washing, the cells were mounted in ProLong Gold Antifade Reagent (Life Technologies) and analyzed. Some sections were subsequently incubated with the proximal tubule marker, fluorescein-conjugated tetragonolobus lotus lectin (1:33, Sigma-Aldrich).^{40 (link)}Six human kidney tissue samples were obtained from the IIS-FJD biobank (age 65–80 years, three males, serum creatinine 0.73–3.1 mg dl⁻¹) with or without AKI. AKI was defined by a serum creatinine increase of 0.3 mg dl⁻¹ within 48 h or by histological evidence of AKI.

Poveda J., Sanz A.B., Carrasco S., Ruiz-Ortega M., Cannata-Ortiz P., Sanchez-Niño M.D, & Ortiz A. (2017). Bcl3: a regulator of NF-κB inducible by TWEAK in acute kidney injury with anti-inflammatory and antiapoptotic properties in tubular cells. Experimental & Molecular Medicine, 49(7), e352-.

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Smad3 and Bcl-3 Protein Expression Analysis

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FLAG-tagged Bcl-3, or HA-tagged Smad3 were cloned into pcDNA3.1 and pcDNA3.0.
All PCR products were confirmed by sequencing. The following antibodies were
used: anti-Bcl-3, c-Myc, cyclinD1, p27, p21, vimentin, E-cadherin,
N-cadherin, ID1, FLAG, HA, ubiquitin (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA, USA), anti-Snail, ID3, RBX1 (Proteintech), anti-phos-ERK,
phos-AKT, AKT, phos-Smad3, Smad3, phos-Smad2, Smad2, Lamin A/C (Cell
Signaling Technology, Boston, MA, USA), anti-GAPDH, donkey anti-Goat IgG
(HRP), goat anti-Mouse IgG (HRP), goat anti-Rabbit IgG (HRP) (KANGCHEN,
Shanghai, China), anti-Actin (Sigma-Aldrich, St. Louis, MO, USA),
anti-K63-ubiquitin, K48-ubiquitin (Millipore, Billerica, MA, USA), donkey
anti-goat coupled to AlexaFluor®488, and donkey anti-mouse or rabbit IgG
coupled to AlexaFluor680 (Invitrogen, Carlsbad, CA, USA). Anti-Smad3
Ser423/425 (Cell Signaling Technology), anti-Smad2/3 pT8,
anti-Smad2/3 pT179, anti-Smad3 pS204, anti-Smad3 pS208, anti-Smad3 pS213
were kind gifts from Dr Liu Fang.

Chen X., Cao X., Sun X., Lei R., Chen P., Zhao Y., Jiang Y., Yin J., Chen R., Ye D., Wang Q., Liu Z., Liu S., Cheng C., Mao J., Hou Y., Wang M., Siebenlist U., Eugene Chin Y., Wang Y., Cao L., Hu G, & Zhang X. (2016). Bcl-3 regulates TGFβ signaling by stabilizing Smad3 during breast cancer pulmonary metastasis. Cell Death & Disease, 7(12), e2508-.

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Immunoprecipitation and Western Blot Analysis

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T_H0 and Treg cells were generated and expanded as described above and 1 × 10⁸ cells were lysed in 4 ml Meister Lysis Buffer (20 mM Tris/HCl pH 7.5, 0.25% NP40, 150 mM NaCl, 1,5 mM MgCl₂ and Protease Inhibitors (Roche, catalogue number: 04693132001) und 1 mM DTT). 60 μl Protein-G beads (Dynabeads Protein G, catalogue number: 10004D) were pre-coupled with 10 μg antibodies (anti-Bcl-3: Santa-Cruz, catalogue number: sc-185; anti-p50: Abcam, catalogue number: ab7971) in PBS and 0.05% Tween and then equilibrated in Meister Lysis Buffer and added to lysed cells and incubated for 4 h. Washing was performed with Lysis Buffer. Proteins were eluted with 80 μl 1 × SDS Lämmli loading dye, one-fourth was used for western blot analysis. Blottings were incubated with anti-p50 (Santa-Cruz, catalogue number: sc-8414) and anti-Bcl-3 antibodies (Santa-Cruz, catalogue number: sc-185).

Reißig S., Tang Y., Nikolaev A., Gerlach K., Wolf C., Davari K., Gallus C., Masri J., Mufazalov I.A., Neurath M.F., Wunderlich F.T., Schattenberg J.M., Galle P.R., Weigmann B., Waisman A., Glasmacher E, & Hövelmeyer N. (2017). Elevated levels of Bcl-3 inhibits Treg development and function resulting in spontaneous colitis. Nature Communications, 8, 15069.

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Immunoblotting and EMSA Assays for Protein Detection

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Immunoblotting was performed using whole cell lysate as previously described (23 (link)). Primary antibodies used include: anti-Bcl3 (Santa Cruz, sc185), anti-p21 (Santa Cruz, sc397), anti-p50 (Santa Cruz, sc7178), anti-GAPDH (Santa Cruz, sc-137179), anti-p53 (Santa Cruz, sc71818), anti-DcR1 (R & D Systems, 398600), anti-HA (Covance, MMS-101R). Alexa-Fluor 680 and Alexa-Fluor 800 fluorescent dye-conjugated secondary antibodies (Invitrogen) were used for visualization with Odyssey Infrared system (LICOR Biosciences). EMSA was performed as previously described (8 (link)) with competition using cold specific and non-specific probes and supershift with anti-p50. The κB probe sequence is shown in Figure 4.

Mansour N.M., Bernal G.M., Wu L., Crawley C.D., Cahill K.E., Voce D.J., Balyasnikova I.V., Zhang W., Spretz R., Nunez L., Larsen G.F., Weichselbaum R.R, & Yamini B. (2015). Decoy receptor DcR1 is induced in a p50/Bcl3-dependent manner and attenuates the efficacy of temozolomide. Cancer research, 75(10), 2039-2048.

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Protein Expression Profiling via Western Blot

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Western blotting was carried out using standard procedures with the following primary antibodies (suppliers): anti-type I collagen (1:1000 dilution, ProteinTech Group, Cat. 14695-1-AP), anti-α-SMA (1:1000 dilution, Abcam, Cat. ab5694), anti-Bcl-3 (1:200 dilution, Santa Cruz Biotechnology, Cat. sc-185), anti-HE4 (1:1000 dilution, ProteinTech Group, Cat. 14406-1-AP), anti-GAPDH (1:3000 dilution, ProteinTech Group, Cat. HRP-60004).

Chen R., Wang L., Liu S., Chen X., Hu Y., Liu H., Zhang H., Jiang Y., Wang Q., Ye D., Li L., Liu D., Pan X., Wei L., Li X, & Zhang X. (2017). Bcl-3 is a novel biomarker of renal fibrosis in chronic kidney disease. Oncotarget, 8(57), 97206-97216.

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Western Blot Analysis of Signaling Proteins

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For Western blot analysis, the following antibodies were used (working dilutions are given in brackets). Rabbit polyclonal antibodies: anti-P(S473)-AKT (1:2000, D9E, Cell Signaling), anti-Bcl-3 (1:1000, C-14, Santa Cruz), anti-P(Thr202, Tyr204)-ERK1/2 and anti-ERK1/2 (both 1:2000, Cell Signaling), anti-ERα (1:2000, Santa Cruz, HC-20), anti-IGF1Rβ (1:2000, Cell Signaling), anti-P(Tyr705)-STAT3 (1:1000, D3A7, Cell Signaling) and anti-STAT3 (1:1000, 79D7, Cell Signaling); rabbit monoclonal antibodies: anti-integrin β1 (1:2000, EPR1040Y, Abcam), anti-GAPDH (1:5000, Ambion) and Ki67 (1: 2000, Epitomics, clone EPR3610); mouse monoclonal antibodies: anti-(pan)AKT (1:1000, 40D4, Cell Signaling), anti-E-cadherin (1:5000, BD Transduction Lab.) and anti-HIF1α (1:1000, BD Transduction Lab.). Anti-CAIX was kindly provided by S. Pastorekova. Secondary antibody conjugates (anti-rabbit/anti-mouse horse radish peroxidase, 1:2000) were purchased from Cell Signaling.
Fulvestrant (LKT Laboratories) was purchased from Biomol (Hamburg/Germany), PQ404 from Calbiochem and recombinant human insulin was from Sigma-Aldrich.

Leyh B., Dittmer A., Lange T., Martens J.W, & Dittmer J. (2015). Stromal cells promote anti-estrogen resistance of breast cancer cells through an insulin-like growth factor binding protein 5 (IGFBP5)/B-cell leukemia/lymphoma 3 (Bcl-3) axis. Oncotarget, 6(36), 39307-39328.

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Western Blot Analysis of Cell Signaling

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Cells were pelleted and solubilized with 0.1% Na-deoxycholate and 0.5% NP-40 in phosphorylation solubilisation buffer (PSB) at 4 °C^{51 (link)}. Whole-cell lysates were subjected to SDS-PAGE and Western blot analysis. The Western blot antibodies anti-BCL3 (rabbit polyclonal, clone C-14, sc-185), anti-GAPDH (mouse monoclonal, clone 6C5, sc-32233), anti-Lamin A/C (goat polyclonal, clone N-18, sc-6215), and anti-p38 (rabbit polyclonal, clone C-20, sc-535) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The antibodies anti-P-p38 (anti-phosphoThr180/phosphoTyr182, mouse monoclonal, clone 28B10, #9216), anti-P-AKT (anti-phosphoSer473, mouse monoclonal, clone 587F11, #4051), anti-TAK1 (rabbit monoclonal, clone D94D7, #5206), anti-P-IκBα (anti-phosphoSer32, mouse monoclonal, clone 14D4, #2859), anti-NFκB p65 (rabbit monoclonal, clone C22B4, #4764), anti-P-IKK-α/-β (anti-phosphoSer176/180, rabbit monoclonal, clone 16A6, #2697), and anti-IκBα (rabbit polyclonal, #9242) were obtained from Cell Signaling Technologies (Danvers, MA, USA) and the antibody anti-p85 (rabbit polyclonal, #06-195) was acquired from Millipore (Billerica, MA, USA). The antibody anti-Tubulin (mouse monoclonal, clone B512, #T5168) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and the antibody anti-NFκB p105/p50 (rabbit polyclonal, ab7971, #GR40002-2) from Abcam (Cambridge, UK).

Poplutz M., Levikova M., Lüscher-Firzlaff J., Lesina M., Algül H., Lüscher B, & Huber M. (2017). Endotoxin tolerance in mast cells, its consequences for IgE-mediated signalling, and the effects of BCL3 deficiency. Scientific Reports, 7, 4534.

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Quantifying NF-κB transcription factors

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In vitro-generated Tregs were restimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin for 2 h and nuclear extracts were prepared. The antibodies used were as follows: anti-Bcl-3: Santa-Cruz (sc-185, 1:1,000 dilution) and Abgent (WA-AP9337c, 1:1,000 dilution), anti-p50: Santa-Cruz, (E-10, 1:1,000 dilution) and Abcam (ab7971, 1:1,000 dilution), anti-p65: Abcam (ab7970, 1:1,000 dilution), anti-p52: Cell Signaling (4882, 1:1,000 dilution), anti-TATA binding protein TBP[EPR3826(2)]: Abcam (ab125009, 1:2,000 dilution) and anti-Actin: Millipore (MAB1501R, 1:5,000 dilution).

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Immunofluorescence Staining of Bcl3 Protein

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Cells were fixed in 4% paraformaldehyde and permeabilized in 0.2% Triton X-100 in phosphate-buffered saline for 10 min, washed in phosphate-buffered saline and incubated overnight at 4 °C with rabbit polyclonal anti-Bcl3 (1:50, Santa Cruz Biotechnology), followed by 1 h incubation with the appropriate FITC secondary antibody (1:300, Sigma-Aldrich). Filamentous actin was stained with phalloidin (1:1000) and cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole. After washing, the cells were mounted in ProLong Gold Antifade Reagent (Life Technologies), and analyzed with a DM-IRB confocal microscope (Leica DM, Bannockburn, IL, USA).

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