Ripa lysis buffer

F-NDP were isolated from extracted carotid arteries by homogenization in RIPA lysis buffer (Teknova Inc., Hollister, CA, USA) at 100 mg/mL. Aliquots (10 µL) of the lysate suspension were applied on the microscope slides and analyzed under LSCM as above under 20× objective.
In animals treated with F-NDP_NV via the femoral artery at high dose, F-NDP_NV were isolated from extracted carotid arteries by solubilizing the clot bearing vessel segment in 12 N hydrochloric acid (Thermo Fisher Scientific) over-night at 60°C at 100 mg/mL.15 The solution was centrifuged (14,000× g at room temperature for 10 minutes), and the pellet was washed once with distilled water. The pellet containing the insoluble F-NDP_NV-Bit was resuspended in DI water while keeping the initial mass/volume ratio. An aliquot of the suspension was applied to a hemocytometer (Incyto Inc., Cheonan, South Korea), which was standardized for particles counting in an inverted fluorescence microscope (Olympus IX81) with 40× objective. Images of F-NDP_NV-Bit were taken from each observation field for counting using TRITC filter cube. Numbers of particles were calculated for the entire solubilized tissue.

Western blot analysis was performed to measure the expression of beclin-1 and LC3-II/I, proteins associated with autophagy (25 (link)). HK-2 cells were lysed using RIPA lysis buffer (Teknova, Inc.) at 4°C for 10–15 min. Protein concentration was determined using bicinchoninic acid assay method. Equal amounts of 50 µg protein extract were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% nonfat milk diluted in Tris-buffered saline containing 0.1% Tween-20 at room temperature for 90 min. The membranes were then immunoblotted for Beclin1 (1:1,000; cat. no. ab55878; Abcam), LC3 (1:1,000; cat. no. ab48394; Abcam), and β-actin (1:1,000; cat. no. ab3280; Abcam) at 20–27°C for 2 h before incubation with secondary antibodies conjugated to horseradish peroxidase-conjugated goat anti-rabbit Immunoglobulin G (1:5,000, cat. no. ab7074; Abcam) at 37°C for 20 min and visualized with enhanced chemiluminescence reagent (Vazyme). Immunoreactive bands were analyzed using Image Pro Plus 6.0 software (Media Cybernetics, Inc.).