Mouse anti gs28

Astrocytes and neurons were lysed with a buffer containing 1% SDS, 2 mM EDTA pH 7.4, 10 mM Tris‐HCl pH 7.4 and proteases inhibitor (1:500). A modified version of the Laemmli buffer was then added to a final 1× concentration (15% SDS, 575 mM sucrose, 325 mM Tris‐HCl pH 6.8, 0.5% β‐mercaptoethanol, 0.01% bromo‐phenol blue). EVs released from 20 × 10⁶ astrocytes were lysed with the same Laemmli buffer. 10 μg of total lysates of neurons or astrocytes and the whole pellet of EVs were loaded on a polyacrylamide gel. Proteins were then separated by electrophoresis, blotted on nitrocellulose membrane and probed using mouse anti‐PrP (1:1000; W226; Dr Lothar Stitz, Institute of Immunology, Tuebingen, Germany), rabbit anti‐Alix (1:500; Covalab, Villeurbanne, France), mouse anti‐Flotillin (1:1000, BD Biosciences, CA, USA), rabbit anti‐Annexin A2 (1:5000, Abcam, UK), rabbit anti‐Tom‐20 (1:500; Santa Cruz Biotechnology, CA, USA), mouse anti‐GS28 (1:1000; BD Biosciences, Franklin Lakes, NJ, USA), mouse anti‐Actin (1:500; Sigma, St. Louis, MO, USA), mouse anti‐βIII‐tubulin (1:4000; Promega, Madison, WI, USA) or Rabbit anti‐GAPDH (1:2000; Synaptic Systems, Gottingen, Germany) antibodies. Photographic development was by chemiluminescence (ECL, Euroclone or FEMTO, Thermo Scientific, MA, USA) according to the manufacturer's instructions. Western blot bands were quantified by ImageJ software.