MM.1S, MM.1R, INA6, RPMI-8226, DOX40, LR5, ANBL6.WT, ANBL6.BR and normal peripheral blood mononuclear cells (PBMCs) were cultured in complete medium. Informed consent was obtained from all patients in accordance with the Helsinki protocol. Bone marrow stromal cells (BMSCs) or plasmacytoid dendritic cells (pDCs) from MM patients were isolated and cultured as described previously.29 (link) Drug source: bAP15, pomalidomide, lenalidomide and bortezomib were purchased from Selleck Chemicals LLC (Houston, TX, USA); and RA190 was obtained from Xcess Biosciences (San Diego, CA, USA). Recombinant human USP14, UCH37 and Rpn11 were purchased from Enzo Life Sciences (Farmingdale, NY, USA), LifeSensors Inc. (Malvern, PA, USA) and Abnova (Taipei City, Taiwan), respectively.
B ap15
Manufactured by Selleck Chemicals
Sourced in United States
B-AP15 is a chemical compound used as a laboratory reagent. It is a proteasome inhibitor that selectively targets the USP14 and UCHL5 deubiquitinases. The core function of B-AP15 is to inhibit the activities of these deubiquitinases.
Automatically generated - may contain errors
Lab products found in correlation
Bortezomib, by Selleck Chemicals (4 mentions)
Dynabeads antibody coupling kit, by Thermo Fisher Scientific (2 mentions)
Anti cyclin d1, by Cell Signaling Technology (2 mentions)
Mts assay celltiter 96 aqueous one solution reagent, by Promega (2 mentions)
Anti gapdh, by Bioworld Technology (2 mentions)
Cytochrome c, by Cell Signaling Technology (2 mentions)
Anti parp, by Cell Signaling Technology (2 mentions)
Mg132, by Selleck Chemicals (2 mentions)
Lenalidomide, by Selleck Chemicals (1 mentions)
Pomalidomide, by Selleck Chemicals (1 mentions)
Etoposide, by Merck Group (1 mentions)
Doxorubicin, by Merck Group (1 mentions)
Pr 619, by Merck Group (1 mentions)
Rabbit anti usp7, by Fortis Life Sciences (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Flavopiridol, by Merck Group (1 mentions)
Mitoxantrone, by Merck Group (1 mentions)
Topotecan, by Merck Group (1 mentions)
Camptothecin, by Selleck Chemicals (1 mentions)
Rabbit anti β actin, by Cell Signaling Technology (1 mentions)
11 protocols using b ap15
1
Evaluating Proteasome Inhibitor Effects on Multiple Myeloma
2
Antibody-based Western Blotting and Immunoprecipitation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following antibodies were used for Western blotting or immunoprecipitation: anti-SF3B1 (Bethyl Laboratories, A300-996A-T, MBL International, D221-3), rabbit anti-cleaved Notch1 (Cell Signaling Technology, 4147S), rabbit anti–β-actin (Cell Signaling Technology, 4967S), rabbit anti-USP7 (Bethyl Laboratories, A300-033A), rabbit anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Cell Signaling Technology, 2118S), rabbit anti-CHEK2 (Bethyl Laboratories, A300-618A), mouse immunoglobulin G2b (IgG2b) (MBL International, M077-3), P5091 (Selleck Chemicals, S7132), PR619 (Sigma-Aldrich, SML0430), b-AP15 (Selleck Chemicals, S4920), ML323 (Sigma-Aldrich, SML1177), BML277 (MedChem Express, HY-13946), BML277 (Sigma-Aldrich, C3742, in vivo), SCH900776 (MedChem Express, HY-15532), camptothecin (Selleck Chemicals, S1288), flavopiridol (Sigma-Aldrich, F3055), topotecan (Sigma-Aldrich, T2705), etoposide (Sigma-Aldrich, E1383), mitoxantrone (European pharmacopoeia reference standard, M2305000), doxorubicin (Sigma-Aldrich, 44583), and E7107 and H3B-8800 (H3 Biomedicine).
3
Investigating b-AP15-Induced Cell Death
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
b-AP15 was obtained from Selleck Chemicals (Houston, TX, USA) and dissolved in DMSO at a stock concentration of 10 mM, aliquoted and stored at −80°C. Other agents are N-acetyl-L-cysteine (NAC, Sigma-AldrichInc., St. Louis, MO); pan caspase Inhibitor Z-VAD-FMK (EnzoLife Sciences International, Inc, Plymouth Meeting, PA). Antibodies (Abs) used in this study were purchased from following sources: anti-CDK2, CDK4, CDK6, anti-cyclinD1, anti-p27, anti-PARP, anti-Bcl-2 (50E3), anti-Bim (Y36), anti-Noxa (D8L7U), anti-BIP (C50B12), anti-CHOP (L63F7), eIF2α, phospho-eIF2α (Ser51), anti-HSP70, anti-HSP90, anti-AIF, cytochrome C and anti-Phospho-MDM2 (Cell Signaling Technology, Beverly, MA, USA); anti-Rb, phospho-Rb (S780), anti-cleaved caspase-8 (Cleaved Asp384) (Assay biotechnology Company, Inc); anti-cleaved caspase-9 p35 (D315), anti-cleaved caspase-3 (p17), anti-AR, anti-MDM2 and anti-GAPDH (Bioworld Technology, Inc). anti-p53(Abcam), anti-ubiquitin (P4D1), anti-K48-linked tetra-ubiquitin, Bax (B-9) (Santa Cruz Biotechnology, Santa Cruz, CA); MTS assay (CellTiter 96 Aqueous One Solution reagent) was purchased from Promega Corporation (Madison, WI, USA). CCK-8 assay kit, PI and Annexin V-FITC apoptosis Detection Kit, DCFH-DA and cell apoptosis Rhodamine 123 Detection Kit were purchased from Keygen Company (Nanjing, China). Dynabeads antibody coupling kit was from Life technologies.
4
Molecular Mechanisms of NF-κB Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
b-AP15 was provided by Selleckchem (United States). LPS (from Escherichia coli, 055: B5), Phorbol 12-myristate 13-acetate (PMA), and Brewer thioglycollate medium were obtained from Sigma–Aldrich (United States). Alexa Fluor 647 phalloidin and primary antibodies were raised against IκBα, ERK1/2, JNK, p38 MAPK, NF-κB P65, PCNA, and GAPDH, and the phosphorylated ERK (Thr202/Tyr204), JNK (Thr183/Tyr185), and p38 MAPK (Thr180/Tyr182) were manufactured by Cell Signaling Technology (United States). The CellTiter 96 Aqueous One Solution reagent used in MTS assays was provided by Promega Corporation (United States). Nuclear and cytoplasmic protein extraction kits were obtained from Keygen Company (China). DAPI was purchased from Abcam (United Kingdom). Enhanced chemiluminescence (ECL) reagents were provided by Santa Cruz Biotechnology (United States). The tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) ELISA kits of mouse and human were obtained from Dakewe Biotechnology (China).
5
Preparation of Chemotherapeutic Agents
6
Generation and Characterization of Plasmid Constructs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmids containing myc-TDP-43 and myc-TDP-43 M337V were generated as described previously [30 (link)]. Plasmids for tau (4R/2N), α-synuclein, USP14 and UCHL5 were purchased from Origene. Mutant plasmids USP14 C114A, USP14 WT ΔUBL, USP14 C114A ΔUBL and UCHL5 C88A were generated from the wildtype plasmid by Genscript. Cells were treated with IU1 (25–100 μM; Selleckchem or Sigma), MG132 (10 μM; Selleckchem), PS341 (10 μM; Selleckchem) and b-AP15 (0.5–2 μM; Selleckchem or Millipore). All compounds were dissolved in DMSO (Sigma), and control samples were treated with the same volume of DMSO.
7
Lung Cancer Cell Line Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All cells were obtained from the American Tissue Culture Collection (ATCC). HCC827, H23, and H1975 cells were used as a model of lung adenocarcinoma (LUAD), while H2170 and HTB-182 cells were used to model lung squamous cell carcinoma (LUSC).
Cells were cultured in RPMI-1940 media (Genesee Scientific, El Cajon, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Genesee Scientific) and 5% penicillin/streptomycin (ThermoFisher Scientific, Waltham, MA, USA) with 5% CO2 at 37 °C. MG132 and b-AP15 were obtained from selleckchem (Munich, Germany).
Cells were cultured in RPMI-1940 media (Genesee Scientific, El Cajon, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Genesee Scientific) and 5% penicillin/streptomycin (ThermoFisher Scientific, Waltham, MA, USA) with 5% CO2 at 37 °C. MG132 and b-AP15 were obtained from selleckchem (Munich, Germany).
8
Analytical Procedures for Organic Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Solvents
were purchased
from commercial sources at high-performance LC (HPLC) or analytical
grade and used without purification. Analytical and preparative LC–MS
were performed using a Waters system equipped with a Waters 2545 Binary
Gradient Module, SecurityGuard ULTRA cartridges for EVO-C18 UHPLC
HPLC, a Kinetex 5 μM EVO C18 100 Å 100 × 3.0 mm column,
and a Waters SQ Detector 2. NMR spectra were recorded using a Bruker
400 MHz spectrometer using the deuterated solvent stated. Chemical
shifts (δ) are quoted in parts per million (ppm) and referenced
to the residual solvent peak. Reagents were purchased from Sigma-Aldrich
unless otherwise stated and used without further purification. b-AP15
and VLX1570 were purchased from Selleckchem and used as supplied without
further purification.
were purchased
from commercial sources at high-performance LC (HPLC) or analytical
grade and used without purification. Analytical and preparative LC–MS
were performed using a Waters system equipped with a Waters 2545 Binary
Gradient Module, SecurityGuard ULTRA cartridges for EVO-C18 UHPLC
HPLC, a Kinetex 5 μM EVO C18 100 Å 100 × 3.0 mm column,
and a Waters SQ Detector 2. NMR spectra were recorded using a Bruker
400 MHz spectrometer using the deuterated solvent stated. Chemical
shifts (δ) are quoted in parts per million (ppm) and referenced
to the residual solvent peak. Reagents were purchased from Sigma-Aldrich
unless otherwise stated and used without further purification. b-AP15
and VLX1570 were purchased from Selleckchem and used as supplied without
further purification.
9
Aumdubin Synthetic Pathway and Experimental Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Aumdubin was synthesized in our laboratory. Other agents used include auranofin (Enzo Life Sciences International Inc.; BML-EI206-0100); bortezomib (S1013), b-AP-15 (S4920) and pan-caspase inhibitor Z-VAD-FMK (S7023) (Selleck Chemicals); N-acetyl-L-cysteine (A7250), tetrathiomolybdate (323446), Rh123 (R8004), Chloro (triphenylphosphine) gold(I) (AuTPP, 254037) (Sigma-Aldrich Inc.); 1-Thio-beta-D-glucose tetraacetate (TG, Alfa Aesar Inc.; L11404.03). Antibodies used in this study were purchased from following sources: antibody against ubiquitin (P4D1) (Santa Cruz Biotechnology; sc-8017); usp1 (8033), usp10 (8501), usp14 (11931), usp15 (66310), bcl-2 (15071), Bax (5023), cyto c (4280), cleaved caspase 9 (20750), caspase 3 (9662), cleaved caspase 3 (9661), PARP (9532), cox4 (4850), and β-actin (4970) (Cell Signaling Technology); Bax used for MEFs (60267-1-Ig), usp2 (10392-1-AP), usp28 (17707-1-AP) (Proteintech Group); uchl5 (Abcam; ab176377); usp30 (Sigma-Aldrich Inc.; SAB4503385); and GAPDH (Bioworld Technology; AP0063).
10
Molecular Signaling Pathway Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The small molecules b‐AP15, ZVAD, imatinib and bortezomib were purchased from Selleck Chemicals and dissolved in DMSO and stored at –20°C. The final dose of DMSO limited to 0.3% in all experiments. The antibodies as follows were obtained from Cell Signaling Technology (Danvers, MA): PARP (#9532S), p27 (#3688S), USP14 (#11931S), Caspase‐3 (#9662S), Caspase‐8 (#9746S), Caspase‐9 (#9508S), XIAP (#2042S), Bax (#5023), Bcl‐2 (15071S), Survivin (#2808S), AIF (#4642S), Cytochrome C (#4272S), PERK (#5683S), ATF4 (#11815S), CHOP (#2895), BCR‐ABL (#2862S), p‐BCR‐ABL (#2861S), STAT5 (#9363S), p‐AKT (#2965), AKT (#4685), CRKL (#3182S) and p‐CRKL (#3181S). The antibodies as follows were obtained from Proteintech (Rosemont, USA): Mcl‐1 (#16225‐1‐Ap) and BCL‐xl (#66020‐I‐Ig). The antibodies as follows are purchased from Abcam (Cambridge, MA): Cleaved Caspase 9 (#AB3629) and Cleaved Caspase 3 (#AB2302). The antibody of ubiquitin was purchased from Santa Cruz Biotechnology. p‐STAT5 (#50095), anti‐mouse (12‐349) and anti‐rabbit (AP132P) antibodies were obtained from Merk Millipore (Darmstadt, Germany). All the above antibodies were used at a dilution of 1:1000.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!