Anti myc tag monoclonal antibody

Chondrocytes, shaved cartilage, and HEK293T cells were minced in SDS buffer. These samples and samples for IP were transferred onto polyvinylidene difluoride (PVDF) membrane after SDS-PAGE. To detect the targeted protein, we used primary antibodies as follows: anti-WWP2 polyclonal antibody (sc-11896, host: goat, Santa Cruz Biotechnology, 1:120), anti-RUNX2 monoclonal antibody (host: rabbit, clone: D1L7F, Cell Signaling Technology, 1:1000), anti-myc tag monoclonal antibody (host: mouse, clone: 9B11, Cell Signaling Technology, 1:1000), anti-myc tag monoclonal antibody (host: rabbit, clone: 71D10, Cell Signaling Technology, 1:1000), anti-DYKDDDK tag monoclonal antibody (host: mouse, clone: 9A3, Cell Signaling Technology, 1:1000), anti-HA tag polyclonal antibody (ab9110, host: rabbit, Abcam, Cambridge, MA, USA, 1:2000) and anti-GAPDH monoclonal antibody (host: rabbit, clone: 14C10, Cell Signaling Technology, 1:1000). Secondary antibody reactions and signal detection were performed using a LI-COR immunofluorescence detection system (LI-COR Biosciences, Lincoln, NE, USA). All uncropped images are provided as a Source Data File.

IP and co-IP experiments were performed using HEK293T cells treated with MG132 (10 μM, 7 h) (Sigma-Aldrich) and ATDC5 cells transduced with retrovirus vector. HEK293T was transfected with plasmid vectors using Lipofectamine 3000 (Life Technologies) according to manufacturer’s instructions. The cells were lysed in lysis buffer (25 mM Tris/HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol). Next, the samples (300 µg per sample) were incubated with primary antibodies (or control IgG), followed by incubation with Dynabeads Protein A or Protein G (Life Technologies) according to the manufacturers’ instructions. ATDC5 cells were transduced using pMXs-myc-Wwp2-transfected PLAT-A cell line (Cell Biolabs, Inc., San Diego, CA, USA) according to the manufacturer’s instructions. After washing with PBS and elution with SDS buffer, western blot analyses were performed as described below. We used the following antibodies: anti-myc tag monoclonal antibody (host: mouse, clone: 9B11, Cell Signaling Technology, 0.1 µg), anti-HA tag high-affinity monoclonal antibody (host: rat, clone 3F10, Sigma-Aldrich, 0.5 ug) and normal mouse IgG2a (Santa Cruz Biotechnology, Dallas, TX, USA, 0.1 µg).