Anti ubiquitin monoclonal antibody

Proteins were fractionated on an SDS-PAGE gel and transferred to an Immobilon-P PVDF membrane (0.45 μm, Millipore). After blocking, membranes were incubated with primary antibodies and then anti-rabbit or anti-mouse secondary antibodies were used for detection. Immunoreactive proteins were visualized using Western Lightening Plus-ECL (PerkinElmer, #NEL105001EA). The following primary antibodies were used: anti-β-actin monoclonal antibody (Sigma, #A5441); anti-USP5 monoclonal antibody (Santa Cruz, #sc-390,943), anti-β-catenin monoclonal antibody (BD Biosciences, #610,153), anti-Slug polyclonal antibody (Santa Cruz, #sc-10,436), anti-E-cadherin monoclonal antibody (BD Biosciences, #610,182), anti-N-cadherin monoclonal antibody (BD Biosciences, #610,921), anti-Vimentin monoclonal antibody (Cell Signaling Technology, #5741S), and anti-Ubiquitin monoclonal antibody (Santa Cruz, #sc-8017).

For the in vivo turnover assay of IAA15 proteins, 10-day-old seedlings overexpressing IAA15^WT or IAA15^P75S were pretreated with 50 μM DEX and 10 μM MG132 for 24 h, and then treated with 10 μM NAA in the presence of 1 mM cycloheximide (CHX) for 4 h. 50 µg total protein samples were separated by SDS-PAGE, and immunoblot analysis was performed using mouse anti-Flag (Sigma, USA). For the ubiquitination assay, 10-day-old DEX-treated IAA15^WT OX and IAA15^P75S OX seedlings were pretreated with 10 μM MG132 for 2 h, and then treated with or without 10 μM NAA for 24 h. Total plant protein (1 mg) was immunoprecipitated using anti-Flag antibody coupled to agarose beads (Sigma, St. Louis, MO, USA) for 4 h at 4°C. The beads were recovered by centrifugation, washed five times with extraction buffer, and eluted with 30 μL SDS-PAGE sample buffer. The eluted samples were separated by SDS-PAGE, and immunoblot analysis was performed using a monoclonal anti-ubiquitin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as described (Miura et al., 2011 (link)).

Experiments were performed as previously described [51 (link)]. The ubiquitination reactions contained 0.1 ug of E1, 0.2 ug of UbE2D2, 5 ug of ubiquitin, and 0.5 ug of E3 (His-tagged MDM2) were performed in a volume of 20 uL for 90 min at 30 °C. Hinokiflavone or MDM2 inhibitor HLI373 (Sigma #373226, Canada) was added to the reaction at the indicated concentrations. Total ubiquitination was detected by immunoblotting using a monoclonal anti-ubiquitin antibody (Santa Cruz, sc-8017). MDM2 ubiquitination was visualized using an MDM2-specific antibody (Santa Cruz, sc-965).