Anti nedd4

Mouse brain homogenates were lysed in RIPA buffer [20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 mM EDTA; 1% NP-40; 1 mM Na₃VO₄ and complete protease inhibitor cocktail (Roche Diagnostics, Barcelona, Spain)] for 20 min at 4°C and centrifuged at 10,000 g for 15 min. Cell culture extracts were prepared using lysis buffer [25 mMTrisHCl pH 7.4, 15 mM EDTA, 50 mM NaF, 0.6 M sucrose, 15 mM Na₄P₂O₇, 1% nonidet P40, 10 mM NaCl, 1 mM PMSF, and a complete protease inhibitor mixture (Roche Diagnostics, Barcelona, Spain)]. Cells were lysed by repeated passage through 24G×5/8″ needle and whole lysates were centrifuged at 10,000 × g for 15 min. Supernatants were collected and 30 μg of total protein subjected to SDS-PAGE, transferred into nitrocellulose blotting membranes (GE Healthcare Life Sciences, Barcelona, Spain) and incubated with the appropriated antibodies: anti-EAAT2 from Santa Cruz Biotech (Barcelona, Spain); anti-Nedd4.2 and anti-Nedd4 from Abcam (Cambridge, UK), anti-actin, anti-HA and anti-Flag (Sigma, Madrid, Spain). After that, they were revealed using goat anti-mouse IRDye 680LT (LI-COR Biosciences, Germany) or goat anti-rabbit IRDye 800CW (LI-COR Biosciences, Germany) and images obtained with the Odyssey Infrared Imaging System (LI-COR Biosciences, Germany). The results were analyzed using the software Image Studio Lite version 3.1 (LI-COR Biosciences, Germany).

Cells were fixed in 4% paraformaldehide for 20 min and subjected to standard indirect immunofluorescence [16 (link)]. Primary antibodies and dilutions were as following: mouse monoclonal anti-Pax7 1:5 and mouse monoclonal anti-MHC (MF20), 1:2 (Developmental Studies Hybridoma Bank, USA),; rabbit polyclonal anti-myogenin M-225 (Santa Cruz Biotechnology, USA), 1:200; chicken anti-Syndecan-4 [74 (link)] 1:500; rabbit polyclonal anti-Nedd4 (Abcam, UK), 1:1000. Secondary antibodies and dilutions were: goat anti-mouse Alexa 594, 1:500; goat anti-rabbit Alexa 488, 1:500; goat anti-mouse Alexa488, 1:500 (Life technologies, USA) and donkey anti-chicken-AMCA (Jackson IR, USA), 1:500. Vectashield (Vector labs, USA) was used for mounting. Images were acquired using an IX71 microscope (Olympus, USA) equipped with a QICam FAST QImaging camera or an Eclipse C2 spectral imaging confocal microscope (Nikon, Japan).