Anti myc m4439

The cells were grown on a cover-glass, washed twice with PBS 1X, and then fixed with 4% paraformaldehyde/PBS for 10 min. Cells were permeabilized with 0.1% Triton X-100 diluted in PBS. Non-specific binding was blocked with 5% bovine serum albumin, 0.05% Tween-20 diluted in PBS for 1 h at room temperature. Cells were incubated with primary antibodies: anti-LRRK2 (1:500 Mjff2 c41-2 Epitomics), anti-Flag (F3165 1:2000 Sigma-Aldrich), anti-Myc (M4439 1:2000, Sigma-Aldrich) diluted in blocking solution, overnight at 4 °C. Cells were then washed with PBS, 0.05% Tween-20 diluted in PBS and incubated with secondary antibodies: Goat anti-Mouse IgG Secondary Antibody Alexa Fluor^® 488 (ThermoFisher Scientific, Waltham, MA, USA) and Goat anti-Mouse IgG Secondary Antibody Alexa Fluor^® 647 (Life Technologies) diluted 1:1000 in blocking solution for 1 h at room temperature. Before analysis, cells were mounted using Mowiol mounting medium, and fluorescence was revealed with a Leica TCS SP5 confocal microscope with LAS lite 170 image software.

HEK293 cells were transfected with the indicated plasmids in 35 mm cell culture plates by LTX/PLUS kit (Thermo Fisher Scientific). At the end of transfection (4 h), the cells were treated or not for 48 h by Edosidin2 (15 µM or 5 µM). The cells were then washed twice in PBS 1X and lysed by 1 mL of NP40 lysis buffer (150 mM NaCl, 1% NP40, 20 mM Tris-HCl pH 7.5, protease inhibitor cocktail). Cellular debris was removed by centrifugation at 13,000× g and cell lysates were pre-cleared by incubation with protein G-agarose beads for 1 h at 4 °C. The protein extracts were then incubated by anti-Flag antibody (F3165, 1:1000, Sigma-Aldrich) or anti-Myc (M4439, 1:1000, Sigma-Aldrich) overnight at 4 °C. After incubation with protein G-agarose for 1 h at 4 °C, the beads were washed four times by lysis buffer. Samples were then eluted in Laemmli buffer and resolved by SDS-PAGE.

For co-immunoprecipitation (Co-IP) assays, HEK293T cells were transfected with Myc-SNAI2 and/or HA-DEAR1 plasmids in a 6 cm plate using Mirus TransIT-LT1 transfection reagent (Mirus, MIR 2300) for 24 h. Cells were treated with MG-132 proteasome inhibitor (Calbiochem, 133407-82-6) for 2 h, before harvest. Cells were lysed with M-PER lysis buffer (ThermoFisher Scientific, 78501) and incubated with pull-down antibody overnight at 4 °C. Then, lysates were incubated with protein A/G agarose beads (Santa Cruz, sc-2003) for 2 h at 4 °C followed by three 5-min washes with RIPA lysis buffer. Proteins were eluted from beads in 30 µL of 2× SDS sample buffer for western blot analysis. Rabbit polyclonal anti-HA (H6908) or anti-Myc (M4439) from Sigma were used for pull-down and Mouse monoclonal anti-HA (H3663) from Sigma or anti-Myc (#562) from MBL International Corporation were used for immunoblotting. For in vivo ubiquitination assays, HEK293T cells were transfected with Myc-SNAI2, 8x-HA-Ub, and/or DEAR1 plasmids in a 10 cm plate. At 20 h after transfection, cells were treated with MG132 20 μmol/L for 4 h and harvested. Cells were lysed with 1× SDS-lysis buffer, denatured by heating at 95 °C for 10 min and then diluted with 0.5% NP-40 buffer and used for further analysis.