Rabbit anti calnexin c4731

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.