Chemidoc chemiluminescence imaging system

Cells were added to a 6-well plate (3×10⁵ cells/well) and incubated for 24 h. Media was then removed and replaced with fresh media containing liposomes. At specified timepoints, the plates were placed on ice, washed once with cold DPBS, and incubated for 15 min with 1 vol% NP40 lysis buffer (50 μL) containing protease and phosphatase inhibitors. The wells were then scraped and the solution added to cold microcentrifuge tubes. Cellular debris was removed by centrifugation (4°C, 5 min). Protein concentrations in samples were determined using the Bradford assay. Equal total protein amounts (~30 μg) were added to each lane of the gel (4–20% gradient). After electrophoresis, the proteins were transferred onto PVDF membranes, blocked with 5% (w/v) milk, and stained with primary antibody overnight. After incubation with the secondary antibody for 1 h, the membrane was incubated with the Western lightning ECL pro substrate (10 min) and then imaged using a ChemiDoc chemiluminescence imaging system (Bio-Rad; Hercules, CA). Western blot images were processed using Image Lab software (Bio-Rad; Hercules, CA). Fiji software was used for densitometry calculations. Loading controls were used as a normalization factor for densitometric calculations.