Anti ulk1 d8h5

Primary antibodies used in Western blot studies included anti-LC3B antibody (#L7543, 1:1000) from Sigma-Aldrich (Burlington, MA, USA), anti-MYC (Y69, #ab32072, 1:1000) from Abcam (Cambridge, MA, USA), anti-ULK1 (D8H5, #8054, 1:1000) from Cell Signaling Technology (Danvers, MA, USA), anti-ULK2 (#PA5-22173, 1:1000) from ThermoFisher Scientific (Waltham, MA, USA), and anti-β-actin (#sc-47778, 1:2000) from Santa Cruz Biotechnology (Santa Cruz, CA, USA). MRT68921 (#S7949), Ara-C (U-19920A, #S1648), and chloroquine (#S6999) were purchased from Selleckchem (Houston, TX, USA). IRDye 800CW goat anti-rabbit IgG (#926-32213, 1:40,000) and anti-mouse IgG (#926-32212, 1:40,000) were used as the secondary antibody (LI-COR Biosciences, Lincoln, NE, USA).

In this study, we utilized MG132 (Z-Leu-Leu-Leu-H (aldehyde), Peptide Institute), bafilomycin A1 (Sigma), and chloroquine (Wako). The antibodies utilized in this study were anti-NRF1 (D5B10; Cell Signaling Technology), anti-p62 (PM045; MBL), anti-S403-P-p62 (4F6; MBL), anti-ULK1 (D8H5; Cell Signaling Technology), anti-TBK1 (ab109735; Abcam), anti-GFP (sc-9996; Santa Cruz), anti-LC3B (L7543; Sigma), anti-GAPDH (6C5; Santa Cruz), anti-α-tubulin (DM1A; Sigma), anti-HA (12CA5; Sigma), and anti-Myc (sc-40; Santa Cruz) for immunoblot analyses; anti-p62 (PM066; MBL), anti-ULK1 (F-4; Santa Cruz), and anti-S172-P-TBK1 (D52C2; Cell Signaling Technology) for immunofluorescence analysis; anti-ubiquitin (clone FK2) (D058-3; MBL), anti-GABARAPL1 (D5R9Y; Cell Signaling Technology) for immunofluorescence and immunoblot analyses, anti-HA (3F10; Sigma) for immunoprecipitation; and normal rabbit anti-IgG antibody (Wako) and rabbit anti-Nrf1 polyclonal antibody (raised against mouse NRF1 residues from 292 to 741)^{33 (link)} for ChIP assay.

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).