Optiprep gradient

Small EV fractions were isolated from right hemibrains according to the method described in (42 (link)) except that the final density gradient was performed using OptiPrep gradient (Sigma-Aldrich) as described in (43 ). After the density gradient centrifugation, eight fractions were collected, rinsed in phosphate buffered saline (PBS), centrifuged at 100,000 × g, and the final pellet of each fraction was suspended in 30 μl of PBS. The protein amount of each fraction was determined using the Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA).

Primary human HSCs were isolated from normal margins of surgically resected liver. Rat HSCs were isolated from normal livers of Sprague-Dawley rats. Mouse HSC were isolated from normal livers of UCHL1^–/– or WT littermate controls. Liver tissue was digested with pronase and collagenase B (Roche) and the cell suspension was subsequently separated by an 11.5% Optiprep gradient (Sigma). HSCs were seeded onto plastic (Corning), cultured in Dulbecco’s modified Eagle’s medium (Life Technologies) supplemented with 16% fetal bovine serum, pyruvate, glutamine, penicillin, and streptomycin (Life Technologies) and maintained in an incubator at 37 °C with 5% CO₂. Freshly isolated (day 0) cells were considered quiescent and (day 10) cultures regarded as activated unless specifically stated otherwise.

Pdia3 exists in plasma membrane caveolae of osteoblasts (9 (link)). To determine the plasma membrane localization of FZD2, FZD5 and ROR2, plasma membranes and caveolae were isolated using a detergent-free method as described previously (44 (link)). Briefly, confluent MC3T3-E1 cultures were harvested by scraping while in isolation buffer (0.25 M sucrose, 1 mM EDTA, 20 mM Tricine, pH 7.8) and were homogenized using a tissue grinder (20 strokes; 10 strokes clockwise and 10 strokes counter-clockwise). Homogenates were centrifuged at 20,000 g for 10 minutes. The supernatant was collected and layered on top of isolation buffer containing 30% Percoll® (GE Healthcare, Piscataway, NJ). The pellet, including nucleus, mitochondria, and endoplasmic reticulum, was discarded. Samples were centrifuged at 84,000 g for 30 minutes. Syringe needles (18G) were used to collect the plasma membrane fraction from the gradient column. The isolated fraction was layered over a 10-20% OptiPrep gradient (Sigma Aldrich, St. Louis, MO), then centrifuged at 52,000 g for another 4 hours. Plasma membrane sub-fractions were collected from the tube, resulting in isolation of thirteen fractions. Caveolae were observed as an opaque band that was collected in fraction 3.