RRMM-BMMNC were treated with different concentrations of LBH589, and 24 h later, total cellular proteins were extracted by using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). The protein samples were separated by using 10% SDS-PAGE gels, and then transferred to a PVDF membrane (Millipore, Bedford, MA). After blocking with 5% non-fat milk, the membranes were then probed with antibodies against Acetyl-H4 (1: 1000, Cell Signaling Technology), PARP (1: 1000, Cell Signaling Technology), and Bcl-x (1: 1000, Cell Signaling Technology). Membranes were washed 3 times and then incubated with horseradish peroxidase (HRP)- conjugated secondary antibodies. The enhanced chemiluminescence detection system was used to visualize the immunoreactive bands.
Bcl x
Manufactured by Cell Signaling Technology
Sourced in United States
Bcl-x is a laboratory reagent used for the detection and quantification of the Bcl-x protein. Bcl-x is a member of the Bcl-2 protein family and plays a role in regulating apoptosis, or programmed cell death.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Merck Group (2 mentions)
Bcl 2, by Cell Signaling Technology (2 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (2 mentions)
Acetyl h4, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Enhanced chemiluminescence reagent, by Cytiva (1 mentions)
Diethyldithiocarbamate, by Merck Group (1 mentions)
Mitochondria isolation kit, by Thermo Fisher Scientific (1 mentions)
Cytochrome c, by Cell Signaling Technology (1 mentions)
Rhodamine 123, by Merck Group (1 mentions)
Survivin, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3 9, by Cell Signaling Technology (1 mentions)
Iap 1, by Cell Signaling Technology (1 mentions)
Iap 2, by Cell Signaling Technology (1 mentions)
Bfl 1, by Cell Signaling Technology (1 mentions)
Annexin 5, by Merck Group (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
Wnt5a b, by Cell Signaling Technology (1 mentions)
5 protocols using bcl x
1
Protein Expression Analysis of RRMM-BMMNC
2
Apoptosis Signaling Pathway Analysis
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GA, diethyl dithiocarbamate (DDC), Annexin V, propidium iodide (PI) and rhodamine-123 were obtained from Sigma-Aldrich (St. Louis, MO). Mitochondria Isolation Kit was obtained from Thermo Scientific (TMO, USA).C9–C10 disrupted GA (GA~) was synthesized by our laboratory19 (link). Antibodies (Abs) against Mcl-1 (S-19), ubiquitin (P4D1), caspase−3, −8, −9, apoptosis-inducing factor (AIF), P27, P21, Bcl-2 and Bax were from Santa Cruz Biotechnology (Santa Cruz, CA). Abs against poly (ADP)-ribose polymerase (PARP, clone 4C10-5) was from BD Biosciences. Abs against p65, phospho-p65 at Ser536, inhibitor of kappa B α (IκBα), phospho-IκBα at Ser32, phospho-Erk1/2 (T202/Y204), Erk1/2, phospho-Akt, Akt, IκB-α, cleaved caspase−3, −9, cytochrome C, Survivin, XIAP, IAP-1, IAP-2, Bcl-x and Bfl-1 were from Cell Signaling Technology (Beverly, MA, USA). Abs against phospho-Stat5A/B (Y694/Y699, clone 8-5-2) and Stat5 were from Upstate Technology; mouse monoclonal antibody against Actin, Cox-4 and PCAN from Sigma-Aldrich. Enhanced chemiluminescence reagents were purchased from Amersham Biosciences (Piscataway, NJ, USA).
3
Bcl-2 Family Protein Expression in Breast Cancer
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The expression of Bcl-2 family proteins in MCF-7 and MCF-7/Adr cells was measured using a western blotting assay (antibodies against Bcl-2, Bcl-x, Bax, and Bid from Cell Signaling, Beverly, MA, USA). Briefly, MCF-7 and MCF-7/Adr cells were separately cultured in an atmosphere containing 5% CO2 at 37°C for 24 h, followed by the addition of free DOX, DOX nanoparticles, targeted DOX nanoparticles or functional DOX nanoparticles for 24 h. The final concentration of DOX was 2 μM. Control experiments were performed by the addition of culture medium alone. Western blot analysis was then performed as described previously [27 (link)].
4
Inflammatory Signaling Pathway Modulation
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Diclofenac and ibuprofen were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and dissolved in dimethyl sulfoxide as 10 mM stock solution, stored at −20 °C. Propidium iodide was purchased from AppliChem (Darmstadt, Germany) and 3,3′-dihexyloxacarbocyanine iodide (DiOC6) from Calbiochem (La Jolla, CA, USA). 4′,6-Diamidino-2-fenilindol (DAPI) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti–rabbit antibodies against p53, Mouse double minute 2 (Mdm-2), AKT, FoxO1, FoxO3a, Fas, caspase-8, caspase-7, caspase-2, RIP, TNF-R2, procaspase-9, procaspase-3, Bcl-2, Mcl-1, Bcl-x, Bax, BimEL, Puma, Bak, Bid, p38, SAPK/JNK, p-SAPK/JNK, Axin, Wnt5a/b, Dvl3, Dvl2, β-catenin, Wnt3a, LRP6, E-cadherin, N-cadherin, vimentin, Snail, Lamin A/C, GAPDH, and β-actin (each 1:500-1:1000) were bought from Cell Signalling Technology (CST, Danvers, MA, USA). Horseradish peroxidase–conjugated secondary anti–rabbit antibodies or anti–mouse antibodies (1:5000) were obtained from CST.
5
Bufalin-induced Apoptosis Pathway
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Bufalin of 99% purity, dimethyl sulfoxide (DMSO), 4′,6-Diamidino-2-Phenylindole, Dilactate, (DAPI), propidium iodide (PI), and Trypsin-EDTA were obtained from Sigma Chemical Co. (St. Louis, MO, USA). McCoy’s 5A medium, fetal bovine serum (FBS), l -glutamine, and penicillin-streptomycin were purchased from GIBCO®/Invitrogen Life Technologies (Carlsbad, CA, USA). Primary antibody anti-β-actin was purchased from Sigma-Aldrich (St. Louis, MO, USA); anti-Bcl-2, Bcl-x, Bax, caspase-3, caspase-8, caspase-9, and PARP were purchased from Cell Signaling Technology (Beverly, MA, USA); anti-cytochrome c, AIF, Endo G, Calpain 1, GRP-78, and GADD153 were purchased from Santa Cruz (Santa Cruz, CA, USA); anti-Fas and caspase-4 were purchased from BD Biosciences (San Diego, CA, USA); and anti-Fas-Ligand was purchased from Millipore (Bedford, MA, USA). Second antibody goat anti-mouse IgG-HRP, goat anti-rabbit IgG-HRP, and goat anti-goat IgG-HRP were purchased from Santa Cruz (Santa Cruz, CA, USA). Second antibody FITC (fluorescein isothiocyanate)-labeled goat anti–mouse IgG Ab, FITC-labeled goat anti-rabbit IgG Ab, and FITC-labeled goat anti-goat IgG Ab were purchased from Jackson ImmunoResearch (West Grove, PA, USA). Bufalin was dissolved in DMSO.
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