Western blot analysis was carried out as previously described (Anker et al., 2018 (link)). For MG132 experiment, cells were treated with 10 μM MG132 (VWR) for 3 hr before 361 was added for another 2 hr treatment, and cells were collected for western blot analysis. For cycloheximide (CHX) chase studies, cells were treated with 361 for 3 hr, then 50 μg/ml of CHX was added, and cells were collected at indicated time points for western blot analysis. Primary antibodies used (see Key Resources Table ): MYC (Y69) (Abcam, ab32072), MYCN (C-19) (Santa Cruz, sc-764), HIF-1α (Novus Biologicals, NB100–134SS), MAX (H-2) (Santa Cruz, sc-8011), Max (S20) (Cell Signaling, 4739S), MYC (phospho T58) (Abcam, ab185655), MYC (phospho S62) (Abcam, ab185656), ANTI-Flag (Sigma-Aldrich, F1804), β-Catenin (BD Bioscience, 610153), Phospho β-Catenin (Ser33/37/Thr41) (Cell Signaling, 9561T), active-β-Catenin (nonphosphorylated) (EMD Millipore, 05–665), Cleaved Caspase-3 (Asp175) (Cell Signaling, 9661S), β-actin (Cell Signaling, 5125S).
Mg132
Manufactured by Avantor
Sourced in Italy
MG132 is a proteasome inhibitor that selectively and reversibly inhibits the chymotrypsin-like activity of the 26S proteasome. It is commonly used in research applications to study the role of the proteasome in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Protein g agarose, by Merck Group (3 mentions)
Sw480, by American Type Culture Collection (2 mentions)
Cycloheximide (chx), by Avantor (2 mentions)
Protein a agarose, by Roche (2 mentions)
M2 conjugated agarose beads, by Merck Group (2 mentions)
Baf155 antibody, by Cell Signaling Technology (2 mentions)
Anti flag, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
β catenin, by BD (1 mentions)
Cleaved caspase 3 asp175, by Cell Signaling Technology (1 mentions)
Phospho β catenin ser33 37 thr41, by Cell Signaling Technology (1 mentions)
Myc y69, by Abcam (1 mentions)
Hif 1α, by Novus Biologicals (1 mentions)
Hct116, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Lipofectamine, by Thermo Fisher Scientific (1 mentions)
Dld 1, by American Type Culture Collection (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
13 protocols using mg132
1
Optimizing Western Blot Analysis for Protein Detection
2
Colorectal Cancer Cell Cultivation
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Human CRC cells (SW480, DLD-1 and HCT116) were obtained from the American Type Culture Collection (Manassas, VA). Isogenic human DLD-1 CRC cell lines expressing either wild-type or mutant K-RAS (D-WT and D-MT cells, respectively) were provided by B. Vogelstein (Johns Hopkins Oncology Center) [15 ]. Cells were propagated at 37 °C and 5% CO2 in DMEM (Gibco) or RPMI 1640 (Gibco) supplemented with 10% FBS (RMBI) and 1% penicillin-streptomycin (Gibco). Lipofectamine (Invitrogen) was used for plasmid transfection, according to the manufacturer’s instructions. ALLN (25 μg/mL; Sigma-Aldrich) and MG132 (20 μM; AMRESCO) were added to media to inhibit protein degradation.
3
Isogenic CRC Cell Lines for EMT Analysis
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HEK293 cells, human CRC cells (LoVo, HCT15, and SW480), and CCD18-Co cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Isogenic human DLD-1 CRC cell lines expressing either WT or MT K-Ras (D-WT and D-MT cells, respectively) were provided by B. Vogelstein (Johns Hopkins OncologyCenter) [29 (link)]. HEK293 reporter cell harboring the chromosomally incorporated TOPflash gene was provided by Dr. S. Oh (Kuk Min University, Seoul, Korea). HEK293 cells, HEK293 reporter cells and LoVo cells were cultured in DMEM containing 10% FBS. CCD18-Co cells were grown in DMEM supplemented with 20% FBS. SW480, HCT15, DLD1 cells were maintained in RPMI1640 medium (Gibco). For EMT experiments, D-WT and D-MT cells were maintained in DMEM containing 10% FBS and EGF (20 ng/mL). L-CM and Wnt3a-CM were prepared as previously described [38 (link)]. Lipofectamine2000 (Invitrogen) was used for plasmid transfection, according to the manufacturer's instructions. ALLN (25 μg/mL sigma-Aldrich), MG132 (20 μM; AMRESCO) were added to media to inhibit protein degradation. All chemicals were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) for the in vitro studies.
4
Plasmid Generation and Reagent Use
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Details of all yeast and human cell line plasmids are listed in Supplementary Data 1 . All plasmids generated in this study were constructed using the Gibson assembly cloning kit (NEB), in accordance with manufacturer guidelines. About 30 bp overlaps were utilized for DNA fragment joining; Sanger sequencing confirmed successful cloning. Chemicals used in this paper: FM4-64 (Thermo Fisher Scientific, T3166), Lucifer yellow (Thermo Fisher Scientific, L453), Thapsigargin (Enzo Life Sciences, BML-PE180-0001), Dynasore (Sigma Aldrich, D7693), MG132 (VWR, 89161-566), CHX (Amresco, 94271-5G), Rapamycin (Fisher Scientific, BP2963-1). ATG5 siRNA was purchased from CST. Short hairpin RNAs were bought from Sigma Aldrich.
5
Immunoprecipitation of Protein Complexes
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For each immunoprecipitation, 12 × 106 cells were treated for 24 h with OHT to activate CRE–ER and 1.5 h with 25 µM MG132 (VWR). Cell lysates were prepared in Kischkel buffer (50 mM Tris⋅HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and Complete Protease Inhibitor mixture [Sigma-Aldrich]), briefly sonicated, and subject to immunoprecipitation overnight with the 12CA5 antibody. Immune complexes were captured with Protein-G agarose (Cell Signaling Technologies), washed in Kischkel buffer, and resolved by SDS/PAGE followed by immunoblotting.
6
Preparation of Plant Growth Regulators
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BAP, GA3 and MeJA were purchased from Sigma-Aldrich (Bornem, Belgium). ABA and ethephon were obtained from Acros Organics (Geel, Belgium). SA, indole acetic acid (IAA) and salt were obtained from Duchefa, whereas mannitol was purchased from VWR (Leuven, Belgium). MG132 was obtained from Enzo Life Sciences (Antwerpen, Belgium). Prior to use, appropriate amounts of MeJA, SA, ABA, IAA and GA3 were dissolved in 100 % (w/v) ethanol, whereas BAP and MG132 were dissolved in 100 % (w/v) dimethyl sulfoxide (DMSO) (VWR). Ethephon, NaCl and mannitol were dissolved in water.
7
Immunoprecipitation of T7 and Flag-Tagged Proteins
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For T7- and Flag-IP experiments, HEK293T cells were transiently transfected for 24 hr with indicated plasmids, along with GFP to tell transfection efficiency, using calcium phosphate transfection. Cells were lysed in Lysis Buffer, sonicated, and debris cleared by centrifugation. Equal amounts of lysates were used for immunoprecipitation with T7 antibody or, in the case of Flag-IP, M2-conjugated agarose beads (Sigma) overnight at 4°C. For T7-IPs, antibody complexes were bound to protein G agarose (Sigma) the next day. Following binding, all IP samples were washed extensively in Lysis Buffer and then boiled in loading dye. For endogenous BAF155 co-immunoprecipitation experiments, samples were processed similarly, except cells were treated for 1.5 hr with 25 μM MG132 (VWR) and then nuclei from cells were extracted in 10mM HEPES, pH 7.9, 10mM KCl, 0.4% NP-40 prior to lysis in Lysis Buffer. Equal amounts of nuclear lysates were subjected to immunoprecipitation with 5 μl of BAF155 antibody (Cell Signaling, 11956) overnight at 4°C and bound to protein A agarose (Roche) the next day. Following four washes as described above, samples were boiled in loading dye.
8
Investigating HeLa Cell Proteasome Inhibition
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The cervical cancer cell line HeLa was purchased from the American Type Culture Collection (ATCC). Cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 μg/ml streptomycin and 100 U/ml penicillin at 37°C under 5% CO2. Cells were plated in 60 mm dishes at a density of 8x105 cells/dish and incubated 4h with 20 μM MG132 (VWR international s.r.l., Milano, Italy) for nuclear extract preparation and ChIP assay, 10 μM lactacystin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) for ChIP assay. As control, cells were treated with the vehicle alone (DMSO, dimethyl sulphoxide) to a final concentration of 0.04% (v/v). In reporter construct experiments, 48h post-transfection cells were incubated with 20 μM MG132 or DMSO for 8h and then harvested for RNA extraction.
9
Protein Turnover Dynamics Regulation
10
Inhibition of PDK1-Akt Signaling Pathway
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PDK1 inhibitor was from Calbiochem (124015). LY294002 was purchased from Cayman Chemical (154447-36-6). U0126 was from Calbiochem (662005). GSK-2141795 was from Activebiochem (A-1504). Pure active Akt enzyme was purchased from SignalChem (A16-10G). Calf intestinal alkaline phosphatase (CIP) was from New England Biolabs (M0290S). MG132 was from VWR International (89161566). Antibodies used were: anti LARP6 antibody from Abnova (H00055323-B01P), anti-HA antibody from Sigma-Aldrich (H9658), anti-collagen α1 (I) antibody from Rockland (600-401-103), anti-collagen α2 (I) antibody from Santa Cruz Biotechnology (sc-8786), anti-fibronectin antibody from BD Transduction Laboratories (610077), anti-β-actin antibody from abcam (ab8227), and anti-pan Akt antibody from Cell Signaling (4691).
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