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3 protocols using lactacystin

1

Antibody Selection and Chemical Inhibitors for Studying Retrovirus Release

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Rabbit polyclonal anti-MuLV p30 antiserum was described previously [13 (link)]. Mouse monoclonal anti-HIV-1 p24CA antibody (YDHIVgp24) was purchased from MyBioSource (San Diego, CA, USA). β-Tubulin was used for the loading control in western blots and was detected by rabbit anti-β-Tubulin antibody (Cell Signaling Technology, Danvers, MA, USA). For western blots, we used an anti-mouse IgG antibody conjugated with horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA, USA) and an anti-rabbit IgG antibody conjugated with horseradish peroxidase (Thermo Fisher Scientific, Waltham, MA, USA). The antibodies for LC3 (Cell Signaling Technology) and p62/SQSTM1 (Proteintech Group, Inc., Rosemont, IL, USA) were used to check autophagic flux in the cells expressing Arf6Q67L. To examine the role of Arf6 in MuLV release, the following chemical inhibitors were used: UNC3230 (MedChemExpress, Monmouth Junction, NJ, USA), ML299 (Aobious Inc., Gloucester, MA, USA), FIPI (Tocris, Minneapolis, MN, USA), LY294002, wortmannin, rapamycin, and bafilomycin A1 (Cayman Chemical, Ann Arbor, MI, USA). MG132 and lactacystin were purchased from AdipoGen Life Sciences (San Diego, CA, USA).
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2

Pharmacological Modulation of Cellular Stress

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We used the glutathione synthesis inhibitor buthionine sulfoximine (25 μM, Cat. no. 309475000, Acros Organics, Fair Lawn, New Jersey), the proteasome inhibitor MG132 (Cat. no. 474790, Calbiochem, EMD Chemicals, San Diego, CA or Cat no. F1100, Ubiquitin-Proteasome Biotechnologies, Aurora, CO), the proteasome inhibtor lactacystin (Cat. no. AG-CN2-0104, AdipoGen, San Diego, CA), the Hsp70/Hsc70 activity inhibitor VER155008 (12.5 μM, Cat. No. 3803, R&D Systems, Inc, Minneapolis, MN), and the heme oxygenase 1 (HO1) inhibitor tin (IV) protoporphyrin IX dichloride (SnPPx, 20 μM, Cat. no. Sn749-9, Frontier Scientific, Logan, Utah).
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3

Inhibitor Screening for Mechano-Signaling

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The protocol followed was the same as described above for RALF1 and mechanical stimulation, but seedlings were pre-incubated with one of the following inhibitors for the indicated amount of time: 2 μM concanamycin A (1 h pre-incubation; BioViotica), 30 μM wortmannin (30 min preincubation; LC Laboratories), 30 μM endosidin 9-17 (30 min pre-incubation; Carbosynth Ltd.), 50 μM MG132 (30 min pre-incubation; Enzo), 25 μM lactacystin (30 min pre-incubation; AdipoGen), 20 μM calpeptin (30 min pre-incubation; Sigma-Aldrich), 20 μM ALLN (30 min pre-incubation; MilliporeSigma), 50 μM Z-VRPR-fmk (30 min pre-incubation; Enzo), 10 μM MALT1 Inhibitor I (30 min pre-incubation; EMD Millipore Corp), and 300 μM LaCl 3 (in phosphate free ¼ MS and 1% sucrose; 15 min pre-incubation; Acros).
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