Mg132

Manufactured by Selleck Chemicals

Sourced in United States, China, Germany, United Kingdom

MG132 is a proteasome inhibitor that reversibly binds to the chymotrypsin-like site of the 26S proteasome. It is commonly used in cell biology research to investigate the role of the ubiquitin-proteasome system in cellular processes.

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Close spelling variants of this product

Proteasome inhibitor MG132 (9 citations) MG132 (S2619) (53 citations)

625 protocols using mg132

In vivo and in vitro phase separation

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For phase separation in vivo, 1,6-hexanediol (Sigma) was added to the medium until the concentration of 1,6-hexanediol was 5%. Cells seeded on coverslips were incubated for 7 min in the medium containing 1,6-hexanediol (as the 1,6-hexanediol incubation group). Some of these cells were then incubated in the medium without 1,6-hexanediol (as the wash out group). MG-132 (Selleck) was added to the medium until the concentration of MG-132 was 5 μM. Cells seeded on coverslips were incubated at 4°C in the medium containing MG-132 for 5 h (as the 4°C group). Some of these cells were then incubated at 37°C for 12 h (as the 37°C group).
For phase separation in vitro, the His-JunB protein was added to the flow cell on the slide, followed by incubation at 37°C for 2 min, then at 4°C for 30 min, and then at 37°C for 2 min. The His-JunB protein was processed at different temperature conditions, and the images were collected.

Ren X., Cui Z., Zhang Q., Su Z., Xu W., Wu J, & Jiang H. (2023). JunB condensation attenuates vascular endothelial damage under hyperglycemic condition. Journal of Molecular Cell Biology, 15(12), mjad072.

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Proteasomal Inhibition in 1205Lu Cells

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1205Lu and 1205LuR cells were seeded in 96-well plates at a density of 7500 cells/ 50 µl per well and left overnight to grow before treatment with 1 µM BI-847325 or 300 nM, 1 µM and 3 µM MG-132 (Selleck) or BI 847325 and MG-132 in combination for 48h. Control cells were treated with vehicle (dimethyl sulfoxide). Proteasomal activity was measured by luminometry using Proteasome-Glo Chymotrypsin-like cell-based assay (Promega) as per the manufacturer’s protocol.

Phadke M.S., Sini P, & Smalley K.S. (2015). The novel ATP-competitive MEK/Aurora kinase inhibitor BI-847325 overcomes acquired BRAF inhibitor resistance through suppression of Mcl-1 and MEK expression. Molecular cancer therapeutics, 14(6), 1354-1364.

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Quantifying Topoisomerase II Cleavage Complexes

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To purify TOP2cc from the cells, we used the rapid approach to DNA adduct recovery (RADAR) assay, as described previously [34 (link)]. Briefly, cells were pretreated with 20 μM MG132 (Selleck Chemicals, S2619) for 1.5 h, and then ETO was added into the medium at a final concentration of 100 μM. After 1 h of incubation, cells were washed with 1× PBS and incubated with a fresh medium containing 20 μM MG132 for the indicated recovery time. Cells were lysed by an MB buffer (6 M guanidinium isothiocyanate, 10 mM Tris-HCl [pH 6.8], 20 mM EDTA, 4% Triton X-100, 1% sarkosyl, and 1% dithiothreitol). DNA was precipitated by adding 100% ethanol, then washed three times in 70% ethanol, and solubilized in 8 mM NaOH. DNA was treated with RNAase A at 37 °C for 1 h and quantified using a NanoDrop spectrophotometer (Thermo Scientific; Waltham, MA, USA). DNA was vacuum transferred to a nitrocellulose membrane using a BioDot SF 246 microfiltration apparatus (BioRad; Hercules, CA, USA) and crosslinked by UVC irradiation. Membranes were analyzed by immunoblotting with the antibodies targeting TOP2 (Abcam, ab52934) for TOP2cc, and dsDNA (Abcam, ab27156) as the loading control. The quantification of TOP2cc was analyzed by measuring the density of the slot blot signal using image J software.

Park J.M., Zhang H., Nie L., Wang C., Huang M., Feng X., Tang M., Chen Z., Xiong Y., Lee N., Li S., Yin L., Hart T, & Chen J. (2023). Genome-Wide CRISPR Screens Reveal ZATT as a Synthetic Lethal Target of TOP2-Poison Etoposide That Can Act in a TDP2-Independent Pathway. International Journal of Molecular Sciences, 24(7), 6545.

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Dissociating R-VEC Tubes for Blotting

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R-VEC vessels were prepared on Matrigel as described above. At the stabilization stage (4 weeks), R-VEC tubes were treated with either 20 μM of MG132 (Selleck Chemicals) or DMSO for 6 h. The medium was removed and the wells were washed once with PBS. R-VEC tubes were then incubated in a solution of 2 mg ml⁻¹ Dispase (Roche) for 45 min at 37 °C to dissociate the tubes. 20μM of MG132 (Selleck Chemicals) or DMSO was continuously provided during the dissociation period. Dissociated cells were collected and further processed for western blotting as described above.

Palikuqi B., Nguyen D.H., Li G., Schreiner R., Pellegata A.F., Liu Y., Redmond D., Geng F., Lin Y., Gómez-Salinero J.M., Yokoyama M., Zumbo P., Zhang T., Kunar B., Witherspoon M., Han T., Tedeschi A.M., Scottoni F., Lipkin S.M., Dow L., Elemento O., Xiang J.Z., Shido K., Spence J.R., Zhou Q.J., Schwartz R.E., De Coppi P., Rabbany S.Y, & Rafii S. (2020). Adaptable haemodynamic endothelial cells for organogenesis and tumorigenesis. Nature, 585(7825), 426-432.

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Ubiquitination of JNK1-GFP in MG-132 treated cells

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To study the role of proteasome degradation, cells were treated with or without MG-132 (Selleckchem, TX, USA) for 5 h. HeLa cells containing ectopically expressed JNK1-GFP were treated with MG-132, and the ubiquitination of immunoprecipitated JNK1-GFP was detected by immunoblot, using an anti-his antibody to detect his-tag-ubiquitin.

Chen H.L., Chiang P.C., Lo C.H., Lo Y.H., Hsu D.K., Chen H.Y, & Liu F.T. (2016). Galectin-7 regulates keratinocyte proliferation and differentiation through JNK-miR-203-p63 signaling. The Journal of investigative dermatology, 136(1), 182-191.

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Modulating Plant Immune Response

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MAPK kinase inhibitor U0126 and proteasome inhibitor MG132 (Selleckchem) were dissolved in DMSO with the stock concentration of 50 mM. After indole priming, leaves were inoculated with spores and inhibitors were added into the green-keeping solution with the final concentration of 100 μM (U0126) or 50 μM (MG132), respectively. Equal amount of DMSO was added as the control. The similar results were obtained from four biological replicated experiments.

Shen Q., Liu L., Wang L, & Wang Q. (2018). Indole primes plant defense against necrotrophic fungal pathogen infection. PLoS ONE, 13(11), e0207607.

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Immunoprecipitation of HA-tagged proteins

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HEK293T cells were transfected with the indicated plasmids. Mg132 (10 μM) was added to the cells for 12 h. Mg132, a proteasome inhibitor, was purchased from Selleck. Two micrograms of HA-TAG antibody were added to the cell lysates and incubated overnight. The next day, protein A/G was added and incubated for 2 h. After that, the mixture was placed on a magnetic stand, the supernatant was discarded, and the magnetic beads or agarose were washed three times. The samples were added to SDS–PAGE loading buffer and examined by immunoblotting.

Zhao Z., Wang H., Kang N., Wang Z., Hou X., Hu L., Qie S., Guo J., Wei S., Ruan X, & Zheng X. (2022). Aurora kinase a promotes the progression of papillary thyroid carcinoma by activating the mTORC2-AKT signalling pathway. Cell & Bioscience, 12, 195.

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Analyzing ER stress in HEK-293T cells

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HEK-293T cells were grown and transiently transfected in 6-well plates, as described above. For experiments with MG-132, cells were pretreated with 10 µM MG-132 (Selleckchem) or DMSO for 1 hour. All cells were treated with 1 µM Thapsigargin or DMSO for 6 hours. Cells were removed from the plate, washed 2x with phosphate buffered saline (PBS), and stained with Zombie Aqua (BioLegend) at 1:1000 in PBS to test for viability. Cells were washed 2x with PBS, fixed with 4% paraformaldehyde, washed once with PBS, washed once with permeabilization buffer (3% (w/vol) BSA, 0.5% saponin (w/vol), and PBS), and incubated in permeabilization buffer for 30 min at room temperature. Cells were spun down and resuspended in permeabilization buffer containing BiP antibody at 1:100 (manufacturer recommended conditions for fixing, permeabilization, and antibody concentrations) for 30 min at room temperature. Cells were washed 3 times with permeabilization buffer and resuspended in permeabilization buffer, with anti-rabbit Alexa-647 (Invitrogen) at 1:1000, for 30 min at room temperature. Cells were washed twice with permeabilization buffer and twice with FACS buffer (1% BSA (w/vol) in PBS) and then resuspended in FACS buffer for analysis by flow cytometry.

Moss S.M., Taylor I.R., Ruggero D., Gestwicki J.E., Shokat K.M, & Mukherjee S. (2019). A Legionella pneumophila kinase phosphorylates the Hsp70 chaperone family to inhibit eukaryotic protein synthesis. Cell host & microbe, 25(3), 454-462.e6.

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Ubiquitination of JNK1-GFP in MG-132 treated cells

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RALF1-mediated 14-3-3 protein degradation

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RALF1 treatment was performed as described by Li et al. (2018) (link) with minor modifications. Eight-day-old seedlings grown on MS were transferred to liquid MS medium containing 1 μM RALF1 for 0.5 h. Medium with an equal volume of buffer was used as a mock control. For MG132 coupled with RALF1 treatment, seedlings of the WT were first treated with 50 μM MG132 (Selleckchem). After 1 h incubation, seedlings were transferred to solution with or without 1 μM RALF1 for 0.5 h. Harvested seedlings were ground in liquid nitrogen for further protein extraction and immunoblotting analysis using anti-Myc and anti-14-3-3 antibodies. For determining whether the degradation of 14-3-3 proteins was dependent on the ubiquitin–proteasome system (UPS), 7-day-old seedlings grown on MS medium were transferred to medium containing 200C/3N with or without 20 μM MG132 for 3 d. Total protein extraction and western blotting were performed as described above.

Xu G., Chen W., Song L., Chen Q., Zhang H., Liao H., Zhao G., Lin F., Zhou H, & Yu F. (2019). FERONIA phosphorylates E3 ubiquitin ligase ATL6 to modulate the stability of 14-3-3 proteins in response to the carbon/nitrogen ratio. Journal of Experimental Botany, 70(21), 6375-6388.

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