Gp62 c

After treatments, cells were fixed and stained using the following antibodies: anti-human p62/SQSTM1 (BD Bioscience; 610832), anti-mouse p62/SQSTM1 guinea pig polyclonal (Progen; GP62-C), LC3B (Cell Signaling; 2775), MTOR-7C10 (Cell Signaling; 2983), ATG13-E1Y9V (Cell Signaling; 13468), and ULK1-D8H5. Cell images were captured by confocal microscopy (Leica; TCS SP5; HCX PL APO CS-63×-1.4-numerical-aperture [NA] objective and HyD GaAsP detection). The number of puncta per cell was determined from confocal scans or directly by epifluorescent imaging, depending on the stain. To detect lysosomal acidification, cells were treated as indicated with 50 nM Lysotracker Red DND-99 (Thermo Fisher; L7528) added during the final 30 min of incubation. The cells were fixed with paraformaldehyde, stored overnight, and imaged by confocal microscopy. The staining intensity was quantified from cytoplasmic regions of interest from multiple cells per field from multiple fields per sample. Quantification of autophagosome puncta and Lysotracker staining were representative of multiple experiments, as detailed in the figure legends.

Western blot experiments were conducted with antibodies raised against LC3 (1:4000; Sigma-Aldrich, #L7543), p62 [1:1000; Enzo Life Sciences, #BML-PW9860-0025; similar data were obtained in pilot experiments when a different anti-p62 antibody was used (1:1000; Progen, Heidelberg, Germany, #GP62-C)], phosphorylated S6K-T389 (1:1000; Cell Signaling, #9206), total S6K (1:1000; Cell Signaling, #9202), phosphorylated ULK1-S757 (1:1000; Cell Signaling, #6888), phosphorylated ULK1-S555 (1:1000; Cell Signaling, #5869), total ULK1 (1:1000; Cell Signaling, #8054) or β-actin (1:4000, Sigma-Aldrich, #A5441), followed by the corresponding HRP-linked secondary antibodies (1:5000; GE-Healthcare, Madrid, Spain, #NA931; GE-Healthcare, #NA934; Invitrogen, #A18769), as appropriate. Densitometric analysis was performed with Image J software (NIH).

Samples were separated using 12% NuPAGE Bis-Tris gels (Invitrogen) in MOPS-SDS buffer, followed by transferring to polyvinylidene difluoride membranes. Antibodies against ubiquitin (Santa Cruz Biotechnology, Inc., P4D1), p62 (Progen Biotechnik, GP62-C), LC3B (Cell Signaling Technology, #2775), GFP (Invitrogen) and Actin (Chemicon International, Inc., MAB1501R) were purchased from the indicated suppliers. Anti-phosphorylated p62 polyclonal antibody was raised in rabbits using the peptide Cys+KEVDP(pS)TGELQSL as an antigen20 (link). The polyclonal antibody against Atg7 was raised in rabbits using the synthetic peptide VVAPGDSTRDRTLDQQ, which corresponds to the amino acid residues 556–571 as an antigen21 (link). For blotting, indicated antibodies were used at a dilution of 1:500. Uncropped blots are shown in Supplementary Fig. 8.