To quantify Aβ42 and apoE uptake, cells were treated, washed with PBS 3 times, then lysed in RIPA buffer (sc-24948 Sigma-Aldrich, St. Louis, MO, USA) supplemented with protease inhibitor cocktail, and phosphatase inhibitor cocktail 2 and 3 (sc-24948 Sigma-Aldrich, St. Louis, MO, USA). Total Aβ42 was measured via ELISA (Thermo Fisher, KHB3544), total apoE was quantified via ELISA (Abcam ab108813).
Uptake was normalized to total mg of protein per sample measured via Bio-Rad DC protein assay kit (5000112 Bio-Rad Laboratories, Hercules, CA, USA).
Cells were plated in 8-well chamber slides (Lab-Tek cc2 plates, 154534PK Thermo Scientific). Cells were washed 3 times with PBS then fixed with 4% paraformaldehyde (Santa Cruz) for 10 minutes at room temperature, washed 3 times with PBS, then permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at room temperature. Cells were blocked with 10% Goat Serum (50062Z Thermo Scientific) for one hour then incubated with goat anti-PGRMC1 (Abcam ab48012 Images were acquired at 40X magnification on a Leica STED 8X Super-resolution Confocal Microscope.
Uptake was normalized to total mg of protein per sample measured via Bio-Rad DC protein assay kit (5000112 Bio-Rad Laboratories, Hercules, CA, USA).
Cells were plated in 8-well chamber slides (Lab-Tek cc2 plates, 154534PK Thermo Scientific). Cells were washed 3 times with PBS then fixed with 4% paraformaldehyde (Santa Cruz) for 10 minutes at room temperature, washed 3 times with PBS, then permeabilized with 0.1% Triton X-100 in PBS for 10 minutes at room temperature. Cells were blocked with 10% Goat Serum (50062Z Thermo Scientific) for one hour then incubated with goat anti-PGRMC1 (Abcam ab48012 Images were acquired at 40X magnification on a Leica STED 8X Super-resolution Confocal Microscope.