Mouse anti alix

The 100,000 × g pellets from N2a NM-HA^sol and N2a NM-HA^agg clones s2E and 1C (adjusted to similar amounts of NM-HA protein, 1:2 and 1:4 dilutions) were resuspended in 2% SDS and loaded onto a preequilibrated nitrocellulose membrane (0.2-µm pore size; Invitrogen) with a Whatman Minifold I dot blotting apparatus (GE Healthcare). Wells were rinsed five times with 200 µl of filter trap assay buffer (1% SDS and 50 mM EDTA in PBS). The membrane was incubated overnight at 4°C with anti-HA 3F10 antibody (1:1,000; Roche) after blocking in 5% skimmed milk for 1 h. For Western blot analysis, protein concentrations were measured by Quick Start Bradford protein assay (Bio-Rad) and proteins were separated on NuPAGENovex 4 to 12% bis-Tris protein gels (Life Technologies) and then transferred onto a polyvinylidene difluoride membrane (GE Healthcare) in a wet blotting chamber. Western blot analysis was performed with mouse anti-Alix (1:1,000; BD Bioscience), rabbit anti-flotillin rbt 3253 (1:1,000; NEB), rabbit anti-Tsg101 ab30871 (1:500; Abcam), rat anti-HA 3F10 (1:1,000; Roche), rabbit anti-calnexin C4731 (1:1,000; Sigma), mouse anti-GAPDH 6C5 (1:5,000; Abcam), and mouse anti-Hsc/Hsp70 N27F3-4 (1:1,000; ENZO) antibodies. The membrane was incubated with Pierce ECL Western blotting substrate (ThermoFisher Scientific) according to the manufacturer’s recommendations.

For Western blot analysis, protein concentrations were measured by Quick Start^TM Bradford Protein assay (Bio-Rad) using the plate reader Fluostar Omega BMG (BMG Labtech) and the corresponding MARS Data Analysis Software (BMG Labtech). Proteins were separated on NuPAGE®Novex® 4–12 % Bis-Tris Protein Gels (Life Technologies) followed by transfer onto a PVDF membrane (GE Healthcare) in a wet blotting chamber. Western blot analysis was performed using mouse anti-LDL receptor (1:500; NovusBiologicals); mouse anti-Alix (1:1000; BD Bioscience); rat anti-HA 3F10 (1:1000; Roche); mouse anti-GAPDH 6C5 (1:5000; Abcam); mouse anti-Hsc/Hsp70 N27F3-4 (1:1000; ENZO); mouse anti-VSV-G 5D4 (1:1000; Sigma); rat anti Sup35 M domain (1:10; hybridoma supernatant);^{88 (link)} rabbit anti-Tau ab64193 (1:1000; Abcam); rabbit anti-Flotillin 1 ab133497 (1:1000; Abcam); mouse anti-SARS-CoV-2-spike S GTX632604 (1:1000; GeneTex); rabbit anti-hACE2 ab15348 (1:1000; Abcam); rabbit anti-clathrin heavy chain (1:1000; Abcam); rabbit anti-GFP (1:5000; Abcam). The membrane was incubated with Pierce^TM ECL Western Blotting Substrate (Thermo Fisher Scientific) according to the manufacturer´s recommendations and imaged with the Imaging system Fusion FX (Vilber Lourmat).