Phospho ser 14 3 3 binding motif

Cells were lysed with a buffer (1% SDS, 100 mM Tris, pH 7.5, 0.5% Triton X-100) containing protease and phosphatase inhibitors. Protein concentration was determined using the Bradford method. Samples (5–10 μg) were separated on 4–12% Bis-Tris gel (NuPAGE®, Life Technologies), and transferred to nitrocellulose membranes (Merk Millipore) for immunoblot analysis using the enhanced chemiluminescence (ECL) method (Dongin LS). Antibodies against the following proteins were used: SQSTM1 (Progen Biotechnik, 1:5000), LC3 (Novus Biologicals, 1:1000), TFEB (Bethyl Laboratories, 1:1000), TFE3 (Sigma Aldrich, 1:1000), phospho-S142-TFEB (Merk Millipore, 1:1000), phospho-(Ser) 14-3-3 binding motif (Cell Signaling Technology, 1:1000), 14-3-3 protein (Cell Signaling Technology, 1:1000), β-actin (ACTB) (Santa Cruz Biotechnology, 1:4000), HA (Cell Signaling Technology, 1:1000) or Lamin A (Santa Cruz Biotechnology, 1:1000). Densitometry of the protein bands was performed using ImageJ.

Cells were solubilized in a lysis buffer containing protease inhibitors. Protein concentration was determined using the Bradford method. Samples (10–30 μg) were separated on 4–12% Bis-Tris gel (NUPAGE, Invitrogen) and transferred to nitrocellulose membranes for immunoblot analysis using the ECL method (Dongin LS). For immunoblotting, Abs against the following proteins were used: Parkin (Santa Cruz, 1:1,000), TFEB (Bethyl Laboratories, 1:1,000), TFE3 (Sigma Aldrich, 1:1,000), phospho-S142-TFEB (Millipore, 1:1,000), phospho-(Ser) 14-3-3 binding motif (Cell Signaling Technology, 1:1,000), HSP70 (Santa Cruz, 1:1,000), Tom20 (Cell Signaling Technology, 1:1,000), HSP90 (Abcam, 1:1,000) and ACTB (Santa Cruz, 1:1,000).

After SDS-PAGE, proteins were transferred to a 0.45 mm polyvinylidene difluoride Immobilon-P membrane (ThermoFisher, IPVH00010) and blocked for 1 h at room temperature (RT) with 5% phosphate-buffered saline-Tween-20 (PBS-T) milk. Membranes were incubated overnight with primary antibodies against the following: LC3 (Novus Biologicals, NB100-2220) 1/3000; β-actin (Abcam, #ab8226) 1/10,000; Map4k3 (Cell Signaling, 9613) 1/1000; RagA (Cell Signaling, 4357) 1/1000; RagC (Cell Signaling, 5466) 1/1000; Lamtor1 (Cell Signaling, 8975) 1/1000; TFEB (Cell Signaling, 4240) 1/1000; pan 14-3-3 (Santa Cruz Biotechnology, sc-629) 1/1000; FLAG (M2) (Sigma; F1804); Raptor (24C12) (Cell Signaling, 2280) 1/1000; phospho-(Ser) 14-3-3 Binding Motif (Cell Signaling, 9601) (for detection of TFEB phosphorylation at Ser211) 1/1000; GST (Santa Cruz Biotechnology, sc-138) 1/1000, in 5% bovine serum albumin in PBS-T. Species-specific secondary antibodies were goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004) or goat anti-mouse IgG-HRP (Santa Cruz, sc-2005), diluted 1/10,000 in 5% PBS-T milk and incubated for 1 h at RT. Chemiluminescent signal detection was captured with Pierce ECL Plus Western Blotting Substrate (ThermoFisher, 321-32) and autoradiography film, using standard techniques. Densitometry analysis was performed using ImageJ. All uncropped scans of immunoblots may be viewed in Supplementary Fig. 9.