Mab1501

Cells were lysed in radio-immunoprecipitation assay buffer (50 mM Tris pH 4.7, 150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, and 1 mM EDTA) with protease and phosphatase inhibitors (Sigma–Aldrich). Cell lysates were quantified using the Pierce BCA protein asay kit (Thermo Fisher Scientific), and proteins were resolved on a 4 to 15% SDS-PAGE gradient gel. Separated proteins were electrophoretically transferred onto polyvinylidene difluoride membranes for subsequent probing with antibodies (CD63 1:1000, ab59479, Abcam; LC3-II 1:1000, CTE4108S, Thermo Fisher Scientific; pan-Actin 1:10000, MAB1501, Abcam). Membranes were washed and incubated with horseradish peroxidase conjugated secondary antibodies (111–035–003, Jackson ImmunoResearch, Pennyslvania, United States). Clarity Western ECL (Bio-Rad, Hercules, California, United States) was used for signal detection using Chemidoc Touch (Bio-Rad). Membranes were stained with Coomassie Blue to observe total protein. Relative protein quantitation was performed by band densitometry using ImageLab 5.2.1 (Bio-Rad); LC3-II was quantified relative to actin, and CD63 relative to total protein after Coomassie staining.