Protease inhibitor cocktail

To obtain whole cell lysates, cells were lysed in a lysis-buffer containing protease inhibitor cocktail (EuroClone, Milan, Italy). Cytoplasmatic and membrane/organelles were isolated using the Cell Fractionation Kit (Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer’s instruction. Proteins were separated on SDS polyacrylamide gel in a Mini-PROTEAN Tetra Cell system (BioRad, Milan, Italy) and transferred to a nitrocellulose membrane using Mini Trans-Blot Turbo RTA system (BioRad, Milan, Italy). Non-specific binding sites were blocked with 5% low-fat dry milk or 5% bovine serum albumin (BSA) in phosphate-buffered saline 0.1% Tween 20 for 1 h at room temperature. Membranes were incubated overnight at 4 °C in primary Abs (anti-TRPML2, anti-VEGFA, anti-VEGFR2, anti-TyR959, anti-TyR996, anti-TyR1059 and anti-TyR1175, anti-NOTCH2, anti-LAMP1, anti-β-actin and anti-GAPDH), followed by the incubation for 1 h at room temperature with HRP-conjugated anti-rabbit or anti-mouse secondary Abs. The detection was performed using the LiteAblot PLUS or Turbo kits (EuroClone, Milan, Italy), and densitometric analysis was carried out by a Chemidoc using the Quantity One software (version 4.6.7, BioRad, Milan, Italy). For quantification, GAPDH and β-actin were used as loading control. One representative out of three independent experiments is shown in each immunoblot.

Cells were lysed in a lysis-buffer containing protease inhibitor cocktail (EuroClone, Milan, Italy). Proteins were separated on 8–14% SDS polyacrylamide gel in a Mini-PROTEAN Tetra Cell System (Bio-Rad, Hercules, CA, USA). Protein transfer was carried out using a Mini Trans-Blot Turbo RTA System (Bio-Rad, Hercules, CA, USA). Membranes previously blocked with 5% low-fat dry milk and 2% bovine serum albumin (BSA) in phosphate-buffered saline 0.1% Tween 20 were incubated overnight at 4°C in primary Abs (anti-TRPML1, anti-LC3, anti-p62, anti-cathepsin B, anti-LAMP1, anti-pERK, anti-ERK, anti-pAKT, anti-AKT, anti-β-actin, and anti-GAPDH), followed by the incubation for 1 h at room temperature with HRP-conjugated anti-rabbit or anti-mouse secondary Abs. The detection was performed using the LiteAblot PLUS or Turbo Kits (EuroClone, Milan, Italy), and densitometric analysis by a Chemidoc using the Quantity One software (version 4.6.7, Bio-Rad, Hercules, CA, USA). GAPDH and β-actin were used as loading controls. One representative out of three independent experiments was shown in each immunoblot figure.