HEK293T cells were transfected with appropriate plasmids. 24 hours later, cells were treated with 10 μM of the proteasome inhibitor MG132 (Calbiochem) for 10 h. Cells were harvested and extracted in 100 µL of co-immunoprecipitation lysis buffer mentioned above supplemented with 1% SDS. Cell extracts were heat-denatured for 5 min at 100°C and diluted with co-immunoprecipitation lysis buffer containing protease inhibitors (Roche) and 20 μM MG132 to an SDS concentration of ≤0.1%. Diluted cell lysates were sonicated and centrifuged to clarify, followed by immunoprecipitation as described above.
Mg132
Manufactured by Roche
Sourced in Germany
MG132 is a laboratory reagent that functions as a proteasome inhibitor. It is commonly used in research to investigate cellular pathways and processes involving protein degradation.
Automatically generated - may contain errors
Lab products found in correlation
Mg132, by Merck Group (5 mentions)
Protease inhibitor cocktail, by Roche (4 mentions)
Mg132, by Enzo Life Sciences (3 mentions)
Protease inhibitor, by Roche (2 mentions)
X tremegene 9, by Roche (2 mentions)
Gfp trap, by Proteintech (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Anti ha antibody conjugated agarose beads, by Roche (1 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
Cleaved notch1 val1744, by Cell Signaling Technology (1 mentions)
Pefabloc, by Roche (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Dynabeads protein g immunoprecipitation kit, by Thermo Fisher Scientific (1 mentions)
Pcmv6 ac gfp sim2, by OriGene (1 mentions)
Anti gfp antibody, by OriGene (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
17 protocols using mg132
1
Proteasome Inhibition in HEK293T Cells
2
Proteomic Analysis of Su(dx) Regulation
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S2 cells were transfected with pMT-N-GFP, pMT-Flag-Ubi (gift from S Bray), and pMT-HA-Su(dx) or pMT-HA-Su(dx)-V5 at 25°C. After 48hr, 1mM CuSO4 was added to induce protein expression, followed by incubation at 18°C for 24hr. The S2 cells were supplemented with 50 μM MG132 (Enzo Life Sciences), 200 μM chloroquine (Sigma), and 10mM NH4Cl at 18°C for 1hr, then either retained at 18°C or transferred to 25°C or 29°C for 15 min. The cells were lysed in lysis buffer (50mM Tris-HCl, pH7.5, 125mM NaCl, 5% glycerol, 0.5% NP-40, 1.5mM MgCl2, 1mM DTT, 1mM EGTA, 1mM N-ethyl-maleimide, 10 μM MG132 and complete protease inhibitor (Roche), and pulled-down with GFP-Trap (ChromoTek).
3
Binding of Vif Mutants to CUL5 Complex
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To determine whether these Vif mutants can bind to CBF-β and other factors in the CUL5 E3 ligase complex, HEK293T cells were transfected with 1 µg of WT or mutant Vif expression vector in six-well plates. To assess the interaction between Vif and A3G or A3F, the ratio of Vif to A3G or A3F was 1∶3 (0.5 µg and 1.5 µg). MG132 (Sigma, cat. no. C2211) at the final concentration of 10 µM was added to the cells 12 h before harvest. Cells were harvested, washed twice with cold PBS and resuspended in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% [v/v] NP-40 and complete protease inhibitor cocktail tablets) at 4°C for 30 min, followed by centrifugation at 13,000 rpm for 30 min. MG132 was dissolved in DMSO and made at the stock concentration of 10 mM. For HA-tag immunoprecipitation, pre-cleared cell lysates were mixed with anti-HA antibody-conjugated agarose beads (Roche) and incubated at 4°C for 3 h. Samples were then washed six times with washing buffer (20 mM Tris-HCl [pH 7.5], with 100 mM NaCl, 0.1 mM EDTA and 0.05% [v/v] Tween-20). The beads were eluted with elution buffer (0.1 mM glycine-HCl, pH 2.0) or 4× loading buffer. The eluted materials were subsequently analyzed by immunoblotting.
4
Notch1 Activation and Immunoprecipitation
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Cells were seeded onto Jagged1-coated plates to activate Notch signaling for 24 h in DMEM-low glucose supplemented with 10% FBS and 1% penicillin/streptomycin. Cells were lysed with 1 mL of ionic buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 0.5% NP40, 0.5% sodium deoxycholate, and 0.05% SDS) supplemented with protease inhibitor cocktail, phosphatase inhibitor, and 10 μM MG132 (all from Roche). Protein A/G Plus-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) and cleaved Notch1 (Val 1744; Cell Signaling Technology) were used for immunoprecipitation. Agarose beads were washed several times with cold Tris buffer containing 1 mM Tris, pH 7.4, and 15 mM NaCl (TBS) and blocked with 3% bovine serum albumin in TBS. Whole lysates were precleared with agarose beads and then incubated overnight (rocking at 4°C) with cleaved Notch1 antibody. Whole cell lysates were added onto new pellets of agarose beads (previously blocked with bovine serum albumin) and incubated for 3 h in a rocking wheel at 4°C. Finally, the supernatant was removed and beads were washed several times with cold 1 × TBS-T (TBS supplemented with 0.05% Tween). Proteins were eluted with 2 × Laemmli buffer supplemented with dithiothreitol and denatured for 5 min at 99°C.
5
Identification of SIM2 Protein Interactors
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TE8 cells were plated at 2.5 × 106 per 10‐cm dish and transiently transfected with either pCMV6‐AC‐GFP‐SIM2 or no insert of pCMV6‐neo (OriGene Technologies) by using Lipofectamine 2000 reagent (Invitrogen). Cells were treated with 5 μmol/L proteasome inhibitor, MG‐132 (Sigma‐Aldrich, St Louis, MO, USA) for 18 hours and, 48 hours after transfection, cells were collected and lysed in lysis buffer (20 mmol/L Tris‐HCl, 20% glycerol, 300 mmol/L KCl, 5 mmol/L 2‐mercaptoethanol, 1 mmol/L Pefabloc (Roche, Basel, Switzerland), and 25 μmol/L MG‐132. Immunoprecipitation was conducted using Immunoprecipitation Kit‐Dynabeads Protein G (Life Technologies, Rockville, MD, USA). Cell lysates were incubated with anti‐GFP antibody (OriGene Technologies) or control mouse IgG at 4°C overnight, and then incubated with 50 μL Dynabeads at 4°C for 4 hours. The Dynabeads‐antibody‐antigen complex was washed and resuspended in SDS sample buffer, then incubated at 95°C for 5 minutes. Eluted proteins were fractionated by SDS‐PAGE and detected by western blot.
6
Proteasome Inhibition in HEK293T Cells
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HEK293T cells were transfected with appropriate plasmids. 24 hours later, cells were treated with 10 μM of the proteasome inhibitor MG132 (Calbiochem) for 10 h. Cells were harvested and extracted in 100 µL of co-immunoprecipitation lysis buffer mentioned above supplemented with 1% SDS. Cell extracts were heat-denatured for 5 min at 100°C and diluted with co-immunoprecipitation lysis buffer containing protease inhibitors (Roche) and 20 μM MG132 to an SDS concentration of ≤0.1%. Diluted cell lysates were sonicated and centrifuged to clarify, followed by immunoprecipitation as described above.
7
Synthesis and Characterization of DCQ
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DCQ was synthesized from 5,6-dichlorobenzofurazan oxide and dibenzoylmethane via the Beirut Reaction [46 (link)]. RPMI 1640, 10X trypsin-EDTA, 0.25X trypsin-EDTA, Dulbecco’s Phosphate Buffered Saline (PBS), Fetal Bovine Serum (FBS), sodium bicarbonate, penicillin-streptomycin (P/S), DMSO, trypan blue, vitamin E, and Dithiothreitol (DTT) were purchased from Sigma, St. Louis, MO, USA. Propidium iodide (PI) and CM-H2DCFDA were purchased from Invitrogen Molecular Probes, Eugene, OR, USA. Protease Inhibitor MG-132 was purchased from Roche Applied Science, Penzberg, Germany.
8
In vivo CENP-A Ubiquitylation Assay
9
Western Blot Analysis of Fly Proteins
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Protein samples isolated from whole adult fly were prepared in lysis buffer (50 mM Tris·HCl, 150 mM NaCl, 10% [vol/vol] glycerol, 1% Triton X-100, 10 mM N-ethylmaleimide, 2 mM EGTA, 1 mM, MgCl2, 50 µM MG-132, and protease inhibitor mixture; Roche), resolved by SDS-PAGE using 12% precast gels (Bio-Rad), and transferred onto nitrocellulose membrane (Bio-Rad) using a Bio-Rad Transblot Transfer Apparatus. Membranes were blocked with 5% skimmed milk in TBS with 0.1% Tween-20 for 1 h at RT and probed with the appropriate primary antibodies diluted in the blocking solution overnight at 4°C. Membranes were washed repeatedly in TBS with 0.1% Tween-20, and then the appropriate HRP-conjugated secondary antibodies (Dako) were incubated for 1 h at RT. Detection was achieved with ECL-Prime detection kit (Amersham). As a loading control, membranes were incubated with anti–α-Tubulin or anti–Actin antibodies for 1 h at RT.
10
Detecting DTX3LM Expression and EMCV 3C Ubiquitination
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For confirmation of DTX3LM expression, HEK293T cells were transfected for 36 h with expression vectors for FLAG-PARP9-c-Myc–DTX3L or FLAG-PARP9M-c-Myc–DTX3LM, then were incubated for 14 h with 20 μM MG-132 (Enzo Life Sciences) and then subjected to lysis and Immunoblot analysis with anti-c-Myc (9E10; Santa Cruz). For analysis of the degradation of EMCV 3C, cDNA encoding EMCV 3C was amplified from EMCV virus stock, its sequence was confirmed by sequencing, then it was tagged with sequence encoding V5 and inserted into the expression vector pcDNA3.1. The plasmid pCI-His-Ub (31815; Addgene) was a gift from A. Winoto (University of California, Berkeley), and plasmid pHA-Ubiquitin (18712; Addgene) was a gift from E. Yeh (University of Texas, Houston). The plasmid encoding TRIM22 was from GeneCopoeia. Cells were transfected with vectors encoding V5-EMCV 3C and His-Ub using X-tremeGene 9 (Roche) and, 36 h later, were treated for 14 h with 20 μM MG-132 (or not) before lysis and Immunoblot analysis. For analysis of the ubiquitination of EMCV 3C, cell lysates were immunoprecipitated with mAb to V5 conjugated to agarose (V5-10; Sigma), followed by elution with 1% SDS, subsequent dilution to 0.1% SDS, reprecipitation with mAb to V5 (identified above), and immunoblot analysis with mAb to HA-tagged ubiquitin (HA-7; Sigma) followed by anti-V5 (identified above).
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