Clone p4d1

We used monoclonal antibodies raised against GFP (clones 7.1 and 13.1; Roche Diagnostics, Meylan, France), HA (clone F7; Santa Cruz Biotechnology, Dallas, TX), anti-ubiquitin antibody coupled to horseradish peroxidase (clone P4D1; Santa Cruz Biotechnology), and polyclonal antibodies against 3-phosphoglycerate kinase (PGK) (clone 22CS; Life Technologies, Saint Aubin, France). Immunoblots were acquired with the LAS-4000 imaging system (Fuji, Tokyo, Japan). Quantification was performed using ImageJ (NIH) on non-saturated blots.

Rabbit polyclonal anti-HSV-1 was purchased from Dako. Chicken anti-β3-tubulin was obtained from Millipore. Mouse monoclonal CHMP4B antibody (clone 13G12) was from Covalab. Antibodies against Lamp1 (clone 1D4B), VPS4 (clone E8), and ubiquitin and polyubiquitinated and ubiquitinated proteins (clone P4D1) were from Santa Cruz Biotechnology.
Secondary antibodies, all Alexa Fluor conjugated, were from Invitrogen: goat anti-rabbit 350 A-21068, goat anti-mouse 555 A-32727, goat anti-chicken IgY 647 A-21449, and goat anti-rat 647 A-21247.

RAW264.7 cells were cultured at an initial density of 4×10⁶ cells per 6-well plate and allowed to adhere overnight. Macrophages were treated with 2.5 µM compound 9 for the indicated period of time. To obtain whole-cell lysates, cells were lysed in cell lysis buffer (10 mM Tris pH 8, 150 mM NaCl, 1% NP-40, 10 mM EDTA pH 8, 1 mM DTT and 1X protease inhibitors), incubated for 15 min on ice and briefly sonicated. Samples were diluted in 5X concentrated sample buffer containing β-mercaptoethanol and denatured at 95°C for 10 min. The samples were separated by SDS-PAGE on an 8% acrylamide gel and transferred to polyvinyldene fluoride membrane (PVDF, Millipore). Immunodetection was performed using an anti-ubiquitin monoclonal antibody (P4D1 clone, Santa Cruz), according to manufacturer’s instructions.

The EphB4 immune-capture antibodies (Santa Cruz) used for coating (0.3 µg/ml) was adsorbed onto 96 well plates (Maxisorb™, Nunc Millipore Sigma) in H₂CO₃/HCO₃- buffer, pH 9.6 o/n at 4 °C followed by non-site blocking in 20 mM Tris pH 7.4, 150 mM NaCl, 0.05% Tween20, 1% bovine serum albumin at 56 °C for 30 min. 10–40 µg cell lysates were incubated for 2 h at 4 °C in presence of protease/phosphatase inhibitors. Primary antibodies incubation was carried out in 50 mM Hepes pH 7.5, 150 mM NaCl, 0.05% Tween20, 1% Bovine Serum Albumin with protease/phosphatase inhibitors incubating for 2 h at 22 °C. Primary monoclonal antibodies raised against either total ubiquitin (Santa Cruz, P4D1 clone, Dallas TX) at 1:200 dilution or K-48 and K-63 branched ubiquitin chains (Cell Signaling, Danvers MA) at 1:100 dilution were used. The ELAST Elisa Amplification System (Perkin Elmer, Waltham MA) was used depending upon the resulting signal intensity. Either OPD (Millipore Sigma, St. Louis MO) or TMB (Thermo Scientific, Waltham, MA) were ELAST Elisa Amplification System (Perkin Elmer, Waltham MA) used as colorimetric substrates for ELISA and reactions ended by adding 2 M sulphuric acid. Optic Density was quantified by spectrophotometric reading at 492 (OPD) or 450 (TMB) wavelengths in a BIOTEK reader (uQuant). All conditions were carried out in triplicates.