Transfections were performed in A375 and Hs294T cells using Lipofectamine RNAi Max (Thermofisher) according to the manufacturer’s protocol. We used siRNA targeting p53 (Santa Cruz Biotechnology), BAX (Cell Signaling), PUMA (Cell Signaling), AURKA (Cell Signaling), p21 (Cell Signaling), Bak (Cell Signaling), BCL-xL (Thermo Fisher), and control non-targeting siRNA (Cell Signaling).
Bcl xl
Manufactured by Thermo Fisher Scientific
Sourced in United States
Bcl-xL is a protein that plays a role in regulating apoptosis, or programmed cell death. It is a member of the Bcl-2 family of proteins and acts as an anti-apoptotic factor, helping to prevent cell death. Bcl-xL is often studied in the context of cancer research and drug development.
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Lab products found in correlation
Bcl2 associated x (bax), by Thermo Fisher Scientific (9 mentions)
Mcl 1, by Thermo Fisher Scientific (6 mentions)
Bcl 2, by Thermo Fisher Scientific (6 mentions)
Gapdh, by Thermo Fisher Scientific (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Cleaved caspase 3, by Thermo Fisher Scientific (3 mentions)
β actin, by Thermo Fisher Scientific (2 mentions)
Bcl xl, by BD (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Taqman universal pcr master mix 4304437, by Thermo Fisher Scientific (2 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (2 mentions)
7900ht real time pcr system, by Thermo Fisher Scientific (2 mentions)
Expression analysis technical manual, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Aurka, by Cell Signaling Technology (1 mentions)
Control non targeting sirna, by Cell Signaling Technology (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Solarbio (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
16 protocols using bcl xl
1
Transfection of Apoptosis-Related Genes
2
Coastal Seaweed Bioactive Compound Extraction
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S. japonica was harvested in Xiapu coastal area, Fujian province, China. Phosphate-buffered saline (PBS) and fetal bovine serum (FBS) were purchased from Solarbio (Beijing, China). The dimethyl sulfoxide (DMSO), DCFH-DA, 1,3-Bis (diphenylphosphino) propane (DPPP), dimethyl sulfoxide (DMSO), diaminofluorophore 4-amino-5-methylamino-2′, 7′-difluorofluorescein diacetate (DAF-FM-DA), bovine serum albumin (BSA), 3-(4, 5)-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), acridine orange, and Hoechst 33342 were purchased from Sigma Co. (St. Louis, MO, USA). Penicillin/streptomycin (P/S) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco (Rockville, MD, USA). Antibodies against GAPDH (clone number of ARC0205, catalog number of MA5-35235), Bax (clone number of ARC0164, catalog number of MA5-35342), Bcl-xL (clone number of C.85.1, catalog number of MA5-15142), and cleaved caspase-3 (clone number of ARC0133, catalog number of MA5-35333) were purchased from Thermo Scientific (Waltham, MA, USA). All other reagents used in this study were of analytical grade and purchased from Solarbio (Beijing, China).
3
Apoptosis Pathway Protein Analysis
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The following antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA): BCL-2 (2876, 1:1000), Caspase-3 (9662, 1:1000) and PARP (9532, 1:1000). The following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): Cytochrome C (7H8: sc-13560, 1:1000), Bcl-xL (H-5: sc-8392, 1:500), Mcl-1 (S-19: sc-819, 1:1000), Bax (N-20: sc-493, 1:500), Bak (G-23: sc-832, 1:500), Bid (FL-195: sc-11423, 1:1000), p53 (DO-1: sc-126, 1:500), and the actin antibody (ACTN05, 1:2000) from Thermo Scientific, (Rockford, IL, USA). Protein concentrations were determined using the Micro bicinchoninic acid (BCA) protein assay (Pierce Chemical Company, Rockford, IL, USA). PNT2258 and associated reagents were provided by ProNAi Therapeutics (Ann Arbor, MI).
4
Melanogenesis Regulation by Tyrosinase
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Mushroom Tyrosinase, α-MSH, dimethyl sulfoxide, MTT, and 2,7-dichlorofluorescein diacetate (DCFH2-DA) were purchased from Sigma Co. (St. Louis, MO, USA). Penicillin/streptomycin (P/S), Dulbecco’s modified Eagle’s medium (DMEM), and fetal bovine serum (FBS) were purchased from Gibco BRL (Life Technologies, Burlington, ON, Canada). Tyrosinase, Bcl-xL, Bax, Tyrosinase-related protein-1 and -2 (TRP-1 and -2), PARP, ERK and p-ERK, cleaved Caspase-3, and β-actin antibodies were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-mouse and anti-rabbit IgG antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). All other chemicals used in this study were analytical grade.
5
Gene Expression Primer and Antibody Protocols
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Primers for HO-1 (Heme Oxygenase-1), Nqo1 (NAD(P)H dehydrogenase quinone 1), Akt (Protein Kinase B), GCLC (Glutamate-cysteine ligase catalytic subunit), GST (Glutathione S-transferase), SOD1 (Super Oxide Dismutase-1), Snxin1 (Sorting nexin-1), Trxr1 (Thioredoxin reductase1), Bcl-xL (B-cell lymphoma-extra-large), and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) are commercially available (Assays-on-Demand Gene Expression Products, Thermo Fisher Scientific, Waltham, MA, USA). Antibodies of Nrf2, HO-1, Akt, and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA), and GCLC and Trxr1 were purchased form Cosmo Bio Co., Ltd. (Tokyo, Japan). SOD1 and Goat anti-rabbit IgG H&L were obtained from Abcam Plc. (Cambridge, UK).
6
Prostate Cancer Cells Sensitization to Paclitaxel
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PC3 or LNCaP cells were seeded at 4 × 105–6 × 105 cells per petri dish (10 cm) for 24 hours in 7 ml RPMI 1640 with 10% fetal bovine serum and grown at 37°C under 5% CO2. Bim (Qiagen), Bcl-xl and MCl-1 (Invitrogen) siRNA was preincubated in 1 ml culture medium without serum before transfection, and then 40 μl transfection reagent (Qiagen) was added to the same culture medium, mixed by vortex and incubated for 5~10 min at room temperature to allow the formation of transfection complexes. The final siRNA concentration was about 5 to 10 nM after adding the complexes drop-wise to PC3 and LNCaP cells. After 24 hours, PC3 and LNCaP cells were treated with 50 nM of paclitaxel and harvested at the indicated time courses.
7
Western Blot Analysis of Apoptosis Markers
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SVF cells and the F4/80-enriched fraction isolated from the liver were collected in lysis buffer containing 20 mM Tris-HCL (pH 8.0), 150 nM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% Nonidet P-40, 2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 0.5 mM PMSF. A modified Lowry protocol was used to quantify protein concentration. Whole AT, hepatocytes, and spleen were sonicated in 500–700 μL of 2% SDS containing 2.5 mM sodium pyrophosphate and 0.5 mM PMSF. Protein was quantified using a bicinchoninic acid (BCA) assay (Thermo Scientific, Waltham, MA). Subsequently, 10–15 μg of protein was electrophoresed through 4–12% Bis-Tris gels (Invitrogen, Grand Island, NY), transferred to a nitrocellulose membrane, and immunoblotted with the following antibodies: cleaved caspase-3, Bax, Bak, Bcl-2, Bcl-xl and phospho-p65. All antibodies were obtained from Cell Signaling Technology (Boston, MA). Blots were developed using either Western Lightning enhanced chemiluminescence substrate and film (Perkin Elmer, Waltham, MA) followed by band intensity quantification using ImageJ64 software, or were imaged using Odyssey Blocking Buffer and the Li-Cor Odyssey Infrared Imaging System (Li-Cor, Lincoln, NE) followed by band intensity quantification using Image Studio Lite Version 3.1 software.
8
Apigenin-Induced Apoptosis and Cell Cycle Arrest
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Apigenin (purity≧99%) was purchased from Extrasynthese (Genay, France); dimethylsulfoxide (DMSO), sodium dodecyl sulphate (SDS), phenylmethylsulfonyl fluoride, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA); the Annexin V-Alexa Fluor 488 and propidium iodide (PI) apoptosis detection kit were purchased from Invitrogen (Molecular Probe, Inc, Eugene, OR, USA). The protein assay kit was obtained from Bio-Rad Labs. (Hercules, CA, USA). Dulbecco’s phosphate-buffer saline (PBS), and trypsin-EDTA were purchased from Gibco-BRL (Gaithersburg, MD, USA). Mouse- or rabbit-monoclonal antibodies specific for cytochrome c, caspase-3, caspase-7, caspase-9, Cyclin B1, Cyclin E, and CDK2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse- or rabbit-monoclonal antibodies specific for phospho-p53, p53, p21, p27, Bcl-2, Bcl-xL, Mcl-1, Bax, Bad, Bak, poly( ADP-ribose) polymerase (PARP), Cdc2, Cdc25C, and Cyclin A were purchased from Invitrogen Corporation (Camarillo, CA, USA). β-Actin antibody was purchased from BD Transduction Laboratories (San Diego, CA, USA). The enhanced chemiluminescence (ECL) kit was purchased from Amersham Life Science (Amersham, UK).
9
Overexpression of STAT1C, Bcl-xL, and Survivin
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FLAG-tagged STAT1C cloned into the backbone of pcDNA3.1 was a gift from Dr. Ouchi (University of New York) [7] (link). Mammalian expression plasmids for pEF-survivin and Bcl-xL was purchased from Addgene. For each experiment, 1×106 ESCC cells were transiently transfected with 10 µg of STAT1C, Bcl-xL or survivin vector or the empty vector (Invitrogen, Burlington, Ontario, CA) in 6-well plates using the lipofectamine 2000 reagent (Invitrogen) as per manufacturer's suggested protocol.
10
Reverse Transfection of Apoptosis Regulators
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Cells were reverse-transfected with 10 nm of BAK (s1880 and s1881), BAX (s1888 and s1889), BIM (s195011), PUMA (pool of siRNAs), BMF (pool of siRNAs), BIK (s1989 and s1990), HRK (s194952), BCL-XL (s1920), MCL-1 (s8583), BCL-w (s1924), BFL-1 (pool of siRNAs) from Life Technologies (Paisley, UK), BID (SI02654568), NOXA (SI00129430), BAD (SI00299348), BCL-2 (S100299411) from Qiagen (Manchester, UK) using Interferin (Polyplus Transfection, NY, USA), according to the manufacturer's protocol and processed 48 h after transfection. Immunoprecipitation and western blotting were carried out according to the standard protocols.26 (link)
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