Mouse anti sqstm1 p62

Immunofluorescence was performed as described [65 (link)], using the following antibodies: goat anti-GFP FITC conjugated (1:400, Abcam), rabbit anti-LC3A/B (1:1000, Cell Signaling), mouse anti- SQSTM1/p62 (1:100, Novus Biological), rabbit anti-TFEB (1:100, Cell Signaling), rabbit anti-CathD (1:100, Calbiochem) and rabbit anti-Atg8a (1:200, Abcam). Anti-rabbit and anti-mouse Alexa Fluor secondary antibodies (Invitrogen) were used at 1:1000. Nuclei were visualized by staining the DNA with DAPI or Hoechst 33324 (Invitrogen). Images were acquired using a Leica TCS SP5 confocal microscope. Live cell images were obtained as previously described [18 ].

Whole flies or HeLa cells were lysed in RIPA buffer containing complete protease inhibitors and phosphatase inhibitors (Roche). Western blots were performed as described previously [18 ]. Antibodies were used at the following concentrations: rabbit anti-P-S6K T398 at 1:1000 (Cell Signaling), guinea pig anti-S6K at 1:10,000 (24), mouse anti-actin at 1:10,000 (Abcam), rabbit anti-LC3A/B at 1:1000 (Cell Signaling), rabbit anti-P-S6K T389 at 1:1000 (Cell Signaling), rabbit anti-S6K at 1:1000 (Cell Signaling), rabbit anti-GAPDH at 1:3000 (Cell Signaling), rabbit anti-P-4E-BP1at 1:1000 (Cell Signaling), rabbit anti-4E-BP1 at 1:1000 (Cell Signaling), rabbit anti-GFP at 1:500 (Cell Signaling), rabbit anti-SQSTM1/p62 at 1:500 (Cell Signaling), Mouse anti- SQSTM1/p62 at 1:1000 (Novus Biologicals), rabbit anti-WDR24 at 1:1000 (Novus Biologicals) and goat anti-Cathepsin D at 1:500 (Santa Cruz). The band intensity was quantified using Image J analysis tool (NIH).