TetR-GFP-tau cells were seeded at 5x10 4 cells per well in a 8-well chamber slide or 6.5x10 5 cells per well in a 6-well plate at day 0 and treated with tetracycline (1 g/l) the following day (day 1) to induce the expression of GFP-tau. Cells were induced for 24 h and treated with MG132 (50 M) (Enzo Life Sciences) or vehicle (DMSO, Sigma) for 4, 6, or 8 h as indicated prior to the end of the induction period. For the transduction of TetR-GFP-tau cells with lentivirus (LV), cells were seeded at 3x10 4 cells per well in a 8-well-chamber slide or 3.5x10 5 cells per well in a 6-well plate on day 0. On day 1, cells were transduced with 10, 20 or 30 multiplicity of infection (MOI) of lentiviral vectors or were untransduced (NT). On day 3, the cells were treated with 1 g/l of tetracycline and the expression of GFP-tau induced for 12-14 h prior to treatment with different drugs. For inhibition of the proteasome, cells were treated with MG132 (50 M) (Enzo Life Sciences) for 8 h. For the autophagy flux assay, cells were treated with MG132 for 8 h, and with bafilomycin A1 (BAF) (100 nM) (Chalbiochem, Millipore) or 3-methyladenine (3-MA) (5 mM) (Sigma) for 3 h prior to the end of the 8 h MG132 treatment.
Mg132
Manufactured by Enzo Life Sciences
Sourced in United States, Germany, Switzerland, France, United Kingdom
MG132 is a proteasome inhibitor. It blocks the activity of the proteasome, a large protein complex responsible for the degradation of many cellular proteins.
Automatically generated - may contain errors
Lab products found in correlation
Cycloheximide (chx), by Merck Group (12 mentions)
Chloroquine, by Merck Group (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Poly d lysine, by Merck Group (3 mentions)
Thapsigargin, by Merck Group (3 mentions)
Mg132, by Roche (3 mentions)
Thapsigargin, by Bio-Techne (3 mentions)
Z vad fmk, by Enzo Life Sciences (3 mentions)
Bi2536, by Selleck Chemicals (3 mentions)
Puromycin, by Merck Group (3 mentions)
Bafilomycin a1, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Bafa1, by Enzo Life Sciences (2 mentions)
Z lle amc, by Merck Group (2 mentions)
H leu amc, by Bachem (2 mentions)
111 protocols using mg132
1
Inducible GFP-tau Expression and Proteasome Inhibition
2
Proteasome and Epigenetic Regulation
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IM9, KMH2, BL2, and Ramos cells were plated (3 × 106 cells per well in a 6-well plate) and treated with the 10 mM proteasome inhibitor MG-132 (Enzo Life Sciences, Farmingdale, NY) for 5 h. Equal amounts of dimethyl sulfoxide were added to the control samples. Cells were lysed and subjected to SDS-PAGE. Western blotting was performed using antibodies against NMT1, NMT2, MCL-1, and GAPDH as the loading controls.
IM9 and BL2 cells were plated at 3 × 106 cells per well in a six-well dish and treated with 1 mM histone acetylation inhibitor suberoylanilide hydroxamic acid (SAHA) and/or increasing concentrations of the DNA methylation inhibitor 5-aza-2’-deoxycytidine (DAC) for 24 h. Equal amounts of dimethyl sulfoxide were added to the control samples. Cells were lysed and subjected to SDS-PAGE. Western blotting was performed using antibodies against NMT1, NMT2, and GAPDH as the loading controls.
IM9 and BL2 cells were plated at 3 × 106 cells per well in a six-well dish and treated with 1 mM histone acetylation inhibitor suberoylanilide hydroxamic acid (SAHA) and/or increasing concentrations of the DNA methylation inhibitor 5-aza-2’-deoxycytidine (DAC) for 24 h. Equal amounts of dimethyl sulfoxide were added to the control samples. Cells were lysed and subjected to SDS-PAGE. Western blotting was performed using antibodies against NMT1, NMT2, and GAPDH as the loading controls.
3
Assess Protein Stability in Transfected HeLa Cells
4
Extracellular Matrix Protein Assay
5
Anti-inflammatory Signaling Pathway Modulation
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The synthetic IKKb inhibitor IMD-0560 was supplied by IMMD Inc. (Tokyo, Japan). Antibodies against cytokeratin 18 (RGE53) and IL-6 (sc-130326) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies against phospho-IKKa/b (16A6), phospho-IkB-a (14D4), IkB-a, NF-kBp65 (D14E12), vimentin (R28), and b-actin were purchased from Cell Signaling Technology (Danvers, MA). Recombinant human TNF-a, IL-1b, LPS from Escherichia coli (serotype 055:B05), and RU486 were purchased from Sigma-Aldrich (St. Louis, MO). Anti-mouse IL-6 receptor (IL-6R) antibody, MR16-1, was kindly provided by Chugai Pharmaceutical Co Ltd (Shizuoka, Japan). MG132 was from Enzo, Life Sciences (Farmingdale, NY). Nonimmune mouse IgG was purchased from Jackson Immuno Research (West Grove, PA).
6
Ubiquitin Immunoprecipitation and Western Blot
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HA-Ubiquitin plasmids were transfected with lipofectamine 3000 reagent in NRK-49F cells, and then the cells were transfected by adenovirus HRD1. After transfection for 24 hours, the proteasome inhibitor MG132 (Enzo Life Science, Ann Arbor, USA) was added to the cells at a nal concentration of 20µM for 6 hours. Then cells were lysed in NP40 lysis buffer. The lysates were centrifuged to gain cytoplasmic proteins and incubated with anti-GSK3β antibody or anti-CK1α antibody overnight, then mixed with protein A/G agarose beads at 4°C for 6 hours. After washing 3 times with NP40 lysis buffer, the beads released proteins by boiling them in 2×SDS loading buffer. The released proteins were analyzed by western blot with anti-ubiquitin antibody (Cell Signaling Technology, Danvers, USA) in 1:1000 dilution.
7
Epidermal Protein Extraction and Lysis
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The fat-free epidermis was immediately placed in liquid nitrogen and pulverized in mortar. The pulverized skin was homogenized on ice for 20 s with a polytron tissue homogenizer and lysed in 1 mL ice-cold lysis buffer [150 mM NaCl, 0.5% Triton-X 100, 50 mM Tris–HCl (pH 7.4), 20 mM ethylene glycol tetra-acetic acid, 1 mM dithiothreitol (DTT), 1 mM Na3VO4 and protease inhibitors, 1 mM phenylmethyl sulfonylfluoride (PMSF), and ethylenediaminetetraacetic acid (EDTA)-free cocktail tablet] followed by a periodical vortex for 30 min at 0 °C. In other studies, MEFs and JB6 C141 cells were incubated with LA, AA (Cayman Chemical Co., Ann Arbor, MI), cycloheximide (Sigma-Aldrich, St. Louis, MO) or MG132 (Enzo Life Sciences, Inc., Farmingdale, NY). The treated cells were harvested, washed with PBS and suspended in the lysis buffer as mentioned above for 1 h on ice. The lysates were centrifuged at 14,800 g for 15 min at 4 °C.
8
Cycloheximide Chase Assay for Protein Turnover
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For the cycloheximide (CHX) chase assay, CHX (Sigma-Aldrich, St Louis, USA) at a concentration of 50 mg/ml was added to the medium; the cells were harvested at the indicated times (0, 1, 2, 4, 6 h) and prepared for Western blotting assays with anti-HA, anti-Flag, and anti-actin antibodies. MG132 (Enzo Life Sciences, Lorrach, Germany) was used as a positive control.
9
NQO1 expression and characterization
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Proteasome inhibitor MG132 was purchased
from Enzo (Farmingdale, NY), and iodixanol solution Visipaque was
from GE Healthcare (Chicago, IL). Other chemicals were from Sigma-Aldrich
(Saint Louis, MO) if not indicated otherwise. Poly-L -lysine
and poly-D -lysine were mixtures of polymers from 1 to 5 kDa
(Sigma P0879 and P0296, respectively). Stock solutions (100 mM) of
the polymers in 50 mM Tris–HCl (pH 7.5) and 500 mM NaCl were
kept at −80 °C.
For mammalian expression of wild-type
(WT) NQO1, 3xFLAG-NQO1 expression construct was used.17 (link) Site-directed mutagenesis was used to clone the read-through
(“no stop”) variants NS1, NS2, and NS3.18 (link) Mutations were verified by sequencing.
The antibody
against FLAG-tag was from Sigma. The antibody against
the TCP-1 ring complexes (TRiC) subunit CCT5 (A303–480A) was
from Bethyl Laboratories (Montgomery, TX). Antibodies against proteasome
subunit PSMC5 (13392), lamin B1 (D9 V6H), histone 2B (D2H6), and GAPDH
(14C10) were from Cell Signaling (Danvers, MA).
from Enzo (Farmingdale, NY), and iodixanol solution Visipaque was
from GE Healthcare (Chicago, IL). Other chemicals were from Sigma-Aldrich
(Saint Louis, MO) if not indicated otherwise. Poly-
and poly-
(Sigma P0879 and P0296, respectively). Stock solutions (100 mM) of
the polymers in 50 mM Tris–HCl (pH 7.5) and 500 mM NaCl were
kept at −80 °C.
For mammalian expression of wild-type
(WT) NQO1, 3xFLAG-NQO1 expression construct was used.17 (link) Site-directed mutagenesis was used to clone the read-through
(“no stop”) variants NS1, NS2, and NS3.18 (link) Mutations were verified by sequencing.
The antibody
against FLAG-tag was from Sigma. The antibody against
the TCP-1 ring complexes (TRiC) subunit CCT5 (A303–480A) was
from Bethyl Laboratories (Montgomery, TX). Antibodies against proteasome
subunit PSMC5 (13392), lamin B1 (D9 V6H), histone 2B (D2H6), and GAPDH
(14C10) were from Cell Signaling (Danvers, MA).
10
Induction of ER Stress and Palmitate Treatment
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Tunicamycin and thapsigargin were purchased from SIGMA, and the stock solution was prepared in dimethyl sulfoxide (DMSO) for the induction of ER stress. Palmitate (SIGMA) was prepared in DMSO then it was diluted to 5 mM with 5% BSA (fatty acid free) (SIGMA)-contained PBS and sonicated at 50 °C until complete dissolution for stock solution. As a negative control of Palmitate, PBS containing 1% DMSO and 5% BSA was used. MG-132 (Enzo Life Sciences, Inc.) was diluted in DMSO and added to medium at final concentration of 5 μM.
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