Anti myc antibody

In vivo ubiquitination assays were performed using 7-day-old WT (Col-0), BES1^OE, BES1^OE/UBP12^OE, and BES1^OE/ubp12-2w/13-3 plants. Seedlings were treated with 1 μM eBL for 6 h. Proteins were extracted with 2× protein extraction buffer as described by Yang et al. (2017) (link) (100 mM Tris–HCl [pH 7.5], 300 mM NaCl, 2 mM EDTA [pH 8.0], 1% Triton X-100, 10% glycerol, 50 μM MG132, and protease inhibitor), and extracts were centrifuged at 12 000 rpm for 10 min at 4°C. After pre-clearing for 1 h, total soluble protein extracts were incubated with TUBE magnetic beads (LifeSensors, cat. no. UM-0402M) at 4°C for 4 h. The magnetic beads were washed 4 times with 1× protein extraction buffer. The pooled protein mixtures were separated by SDS–PAGE and analyzed by immunoblotting with anti-myc antibody (Proteintech, cat. no. 16286-1-AP) and anti-Ubi antibody (Santa Cruz Biotechnology, P4D1, cat. no. sc-8017). All assays were repeated in three independent experiments.

HEK293T cells were transfected with 3XFLAG-AnkH, BAP, and c-myc-LARP7 for 24 h and collected in lysis buffer, as described previously (79 (link), 99 (link)). FLAG-tagged and myc-tagged proteins were immunoprecipitated by using anti-FLAG M2 magnetic beads (Sigma) or SureBeads protein G magnetic beads (Bio-Rad) cross-linked with anti-myc antibody (Proteintech).

The cell lysates were incubated with the indicated antibodies overnight at 4 °C with gentle mixing, and then precipitated with Protein A+G agarose beads (Santa Cruz Biotechnology, Dallas, TX, USA) for 3 h at 4 °C. The beads were then washed five times with lysis buffer and eluted with 2× SDS sample buffer. Western blot analysis was then performed. The primary antibodies used for immunoprecipitation were as follows: anti-Flag antibody (Applied Biological Materials, Richmond, BC, Canada; 5 μg/mL), anti-HA antibody (Cell Signaling Technology, Beverly, MA, USA; 5 μg/mL), anti-Snail antibody (Cell Signaling Technology, Beverly, MA, USA; 5 μg/mL), and anti-Myc antibody (Proteintech; 5 μg/mL).