For total protein extraction, Arabidopsis seedlings were harvested and ground using a Mini-Beadbeater-24 (BioSpec Products, Inc.) in three volumes (mg/µL) of extraction buffer containing 100 mM Tris-Cl pH 7.5, 100 mM NaCl, 5 mM EDTA, 1% SDS, 5 mM DTT, 10 mM β-mercaptoethanol, 40 µM MG115 (Sigma-Aldrich), 40 µM MG132 (Sigma-Aldrich), 1× phosphatase inhibitor cocktail 3 (Sigma-Aldrich), 1× EDTA-free protease inhibitor cocktail (Roche), and 0.01% bromophenol blue. Samples were immediately boiled for 10 min and centrifuged at 16,000×g for 10 min. Proteins in the supernatant were separated by SDS-PAGE, transferred to nitrocellulose membranes, probed with the indicated primary antibodies, and then incubated with horseradish peroxidase-conjugated anti-goat, anti-mouse, or anti-rabbit secondary antibodies (Bio-Rad). Primary antibodies, including monoclonal mouse anti-HA antibodies (Sigma-Aldrich, H3663), polyclonal goat anti-HA antibodies (Genscript, A00168), polyclonal rabbit anti-HMR antibodies (homemade), polyclonal rabbit anti-PIF4 antibodies (Agrisera, AS 12 1860), and polyclonal rabbit anti-RPN6 antibodies (Enzo Life Sciences, BML-PW8370-0100) were used at 1:1000 dilution. Signals were detected by chemiluminescence using a SuperSignal kit (ThermoFisher Scientific).
Mg115
Manufactured by Merck Group
Sourced in United States
The MG115 is a laboratory centrifuge designed for general purpose applications. It features a compact and robust construction, with a maximum speed of 6000 RPM and a maximum RCF of 4,220 x g. The unit is equipped with a removable rotor that can accommodate various sample tube sizes.
Automatically generated - may contain errors
Lab products found in correlation
Mg132, by Merck Group (5 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions)
Keratinocyte sfm media, by Thermo Fisher Scientific (2 mentions)
Plasmocin prophylaxis, by InvivoGen (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
High glucose dmem, by Thermo Fisher Scientific (2 mentions)
Phosphatase inhibitor cocktail 3, by Merck Group (1 mentions)
Mini beadbeater 24, by Biospec (1 mentions)
Mouse monoclonal anti ha antibody, by Merck Group (1 mentions)
Edta free protease inhibitor cocktail, by Roche (1 mentions)
Lactacystin, by Merck Group (1 mentions)
Monosodium phosphate, by Merck Group (1 mentions)
Gliotoxin, by Merck Group (1 mentions)
Phenylmethylsulphonyl fluoride, by Merck Group (1 mentions)
Sorbitol, by Merck Group (1 mentions)
Albumax 2, by Thermo Fisher Scientific (1 mentions)
Pr 619, by Merck Group (1 mentions)
Glutathione acceptor beads, by PerkinElmer (1 mentions)
Protein a donor beads, by PerkinElmer (1 mentions)
Complete mini edta protease inhibitor cocktail, by Roche (1 mentions)
9 protocols using mg115
1
Total Protein Extraction from Arabidopsis
2
Comprehensive Ubiquitin Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hypoxanthine, sorbitol, percoll, MG132, Epoxomicin, Lactacystin, Clasto-Lactacystin β-lactone, Gliotoxin, MG115, EDTA, sodium phosphate dibasic, monosodium phosphate, Tris HCl, sodium chloride, Tween-20, sodium pyrophosphate, glycerol-2-phosphate, saponin, bovine serum albumin (BSA), dithiothreitol (DTT) and phenylmethylsulphonyl fluoride (PMSF) were purchased from Sigma. Glutathione acceptor beads, protein A donor beads, DELFIA enhancement solution and DELFIA secondary antibody (Eu-N1 rabbit Anti-mouse-IgG) came from Perkin Elmer. NP-40 was purchased from Calbiochem, sodium fluoride from Panreac, antibody anti-ubiquitin P4D1 from Santacruz, deubiquitylases inhibitor PR-619 came from Merck, complete mini EDTA protease inhibitor cocktail from Roche, antibody anti-ubiquitin FK2 from Enzo, biotin-TUBEs from Life sensor, RPMI 1640 medium from Gibco, AlbuMAX II from Invitrogen, bortezomib from Selleckchem, enhanced chemiluminescence (ECL) from GE Healthcare and PBS from Oxoid. Atovaquone was prepared in house.
3
Analyzing SOCS1 Regulation of HIV-1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293T cells (3.0×105 cells/well in a 12-well plate) were co-transfected with 0.1 µg of pNL4-3, 0.3 µg of pRhTRIM5α-HA and 0.6 µg of pHuSOCS1 using FuGENE6 (Promega, Madison, WI) according to the manufacturer's instructions. The total amount of plasmids transfected was adjusted to 1.0 µg per sample with pcDNA3.1. For experiments with increasing amounts of pHuSOCS1 in the presence or absence of HIV-1, HEK293T cells were transfected with 0.1 µg of pUC18 or pNL4-3, 0, 0.0375, 0.075, 0.15, 0.3 or 0.6 µg of pHuSOCS1 and with or without 0.3 µg of pRhTRIM5α-HA. The total amount of plasmids was adjusted to 1.0 µg per sample with pcDNA3.1. To block proteasome-dependent protein degradation, cells were treated with 30 µM of MG115 (Sigma-Aldrich) or 30 µM MG132 (Sigma-Aldrich) for 16 hours.
4
Engineered Antigen Presenting Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lines expressing HLA‐A2 alone or coexpressing HLA‐A2 and the human costimulatory ligands CD80, CD86, 4‐1BBL, or CD70 were generated by retroviral transduction of the K562 cell line, a human erythroleukemia cell line (ATCC® CCL‐243, Manssas, VA), in conjunction with flow sorting. To generate eAPC or eAPC expressing costimulatory ligands, K562 cell clones with high levels of expression of these molecules were selected and were transduced with a retroviral construct encoding five major HLA‐A2 restricted antigenic peptides (GILGFVFTL‐Influenza M; FMYSDFHFI‐Influenza A; CLGGLLTMV‐EBV‐LMP2A; GLCTLVAML‐EBV‐BMLF1; NLVPMVATV‐HCMVpp65) linked via a GGS3 peptide linker to eCFP and a PEST sequence [47 ] for efficient proteasome targeting and antigen processing. To analyze the proteasome targeting of this construct, eAPC were treated for 16 h with proteasome inhibitor MG115 (1 μM; Sigma Aldrich) and analyzed for eCFP expression by flow cytometry.
5
Cell Culture Conditions for Prostate Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RWPE1, LNCaP, Phoenix A, and 293T cells were obtained from ATCC. LNCaP-Abl (passage 75), 22Rv1, DU145 and PC3 cells were kind gifts from Dr. Zoran Cullig (University of Innsbruck, Austria), Dr. Michael Henry (University of Iowa), and Dr. Frederick Domann (University of Iowa), respectively. RWPE-1 cells were maintained in Keratinocyte-SFM media supplemented with 5 ng/ml epidermal growth factor (EGF) and 0.05 ng/ml bovine pituitary extract (BPE; Life Technologies). LNCaP and 22Rv1 cells were maintained in RPMI 1640 medium. DU145, Phoenix A and 293T cells were maintained in high glucose DMEM (Life Technologies). PC3 cells were maintained in DMEM-F12 (Life Technologies). All three media contained L-glutamine, 10% Fetal Bovine Serum (FBS), 100 units/ml Penicillin, 100 μg/ml Streptomycin, 1 mM sodium pyruvate and 15 μg/ml Plasmocin™ prophylaxis (InvivoGen). LNCaP-Abl cells were cultured in RPMI medium complemented with 10% charcoal stripped FBS (CS-FBS). A humidified hypoxia chamber calibrated to 1% O2 using a 95%N2/5%CO2 gas mixture (Coy Labs) was used for hypoxia treatment. Cells were seeded at a density of 142 cells/mm2 and 38 cells/mm2 for short (< 96 h) and long (6 days) hypoxia treatments, respectively. Proteasome inhibitors MG132 and MG115 (Sigma) were applied to a final concentration of 25 μM.
6
Analyzing NPR1-GFP Protein Interactions
7
Inhibiting Cellular Pathways in Neutrophil Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For inhibition experiments, cells were preincubated in the presence of 25 ng/mL GM‐CSF for 30 minutes with the following inhibitors: diphenyleneiodonium (DPI) 20 µM, dinitrophenol (DNP) 750 µM, cytochalasin D 10 µg/mL, oligomycin 10 µM, 2‐deoxy‐D‐glucose 2 mM, EDTA 5 mM, rotenone 10 µM, antimycin 5 µM, ciclosporin 0,5 ng/mL, leupeptin 20 µM, MG‐115 1 µM (all Sigma‐Aldrich, St. Louis, MO, USA), RO‐31‐8220 100 nM, SB203580 10 µM, R406 1 µM, rapamycin 100 nM, wortmannin and chloroquine 10 nM, Nec‐1s 50 µM (all Selleckchem, Houston, TX, USA), bafilomycin A 500 nM, GSK484 1 mM, chymostatin 1 µM, z‐VAD 10 µM, GSK872 100 nM (Cayman Chemicals, Ann Arbor, MI, USA) LDC7559 1 µM (MedChemExpress, NJ, USA), and necrosulfonamid 5 µM (Merck Millipore, Burlington, MA, USA). These concentrations had been optimized in pilot‐experiments. Inhibitors by themselves did not induce DNA‐release from neutrophils (data not shown).
8
Cellular Stress Response Modulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For low temperature treatment, cells were incubated at 30 °C under 5% CO2 for the indicated times. For chemical treatment, cells were incubated with the following reagents at the indicated final concentrations in the culture media. Proteasome inhibitors (9 μm MG132 and MG115, Sigma-Aldrich), 10 μm pepstatin A (Sigma-Aldrich), and a series of different concentrations of UBEI-41 (Biogenova) were incubated with cells for 16 h at 37 °C; 0–2 mm sodium 4-phenylbutyrate (PBA) and 0–0.4 m glycerol were incubated with cells for 30 h at 37 or 30 °C.
9
Cell Culture Conditions for Prostate Cancer
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!