Rapa-Induced Macrophage Autophagy Evaluation

To evaluate the activity of Rapa, free or loaded into Nano and KP-Nano, macrophages were treated with the same amount of Rapa (free or entrapped into the particles). In particular, RAW264.7 cells were seeded at 2.5 × 10⁴ cells/cm² and treated the day after with Rapa 130 nM, free or loaded into Nano or KP-Nano. Cells were lysed at 24 h after treatments and SDS-PAGE and Western blotting were performed according to standard protocols and as described in [33 (link)]. Briefly, 20 μg of proteins obtained from each condition were loaded onto Bolt Bis-Tris gel 4–12% (ThermoFisher Scientific, Waltham, MA, USA) and transferred on nitrocellulose membranes (GE Healthcare; Chicago, IL, USA). Blocking was done in a 5% BSA solution (5% BSA, 20 mM Tris, 140 mM NaCl, 0.1% Tween-20). The following primary antibodies were used for the staining: anti-Phospho-ULK1 (Ser757) (1:500, cat. Number 14202 Cell Signaling Technology, Danvers, MA, USA), anti- ULK1 (D8H5) (1:500, cat. Number 8054 Cell Signaling Technology, Danvers, MA, USA), and anti-α-tubulin (1:1000, cat. number sc398103, Santa Cruz Biotechnology. Dallas, TX, USA). All secondary antibodies were obtained from Thermo Fisher Scientific. The chemiluminescence signal was detected using the ChemiDoc Biorad acquisition instrument and obtained images were analyzed with the Image Lab software (Bio-Rad; Hercules, CA, USA).