α lc3b

For Immunofluorescence studies, cells were grown on glass coverslips for 2 days, then the coverslips were washed once in PBS and then fixed with 4% p-formaldehyde for 30 min at room temperature. Immunofluorescence staining of cells was carried out as previously described.^{39 (link)} An Olympus FV1000 confocal microscope with a 60 × objective and a Zeiss 710 confocal microscope with × 63 objective were used for taking images.
Antibodies used were as follows: α-Myc-Tag (Millipore; 4A6), α-Myc-Tag (Cell Signaling; #2272), α-LC3B (Cell Signaling; #2775), α-LAMP2 (BD Pharmingen, San Jose, CA, USA; CD107b), α-EEA1 (Abcam, ab2900), α-Calnexin (Cell Signaling; #2679), α-WIPI2 (Bio-rad; MCA578OGA) and α-GM130 (BD Bioscience, #610822).
For live-cell imaging, cells were transfected with GFP-tagged DRAM-3 and RFP-tagged Rab5 or Rab7 at least 2 days before confocal analysis. An Andor spinning disc confocal system with Cairn Optsplit (for simultaneous GFP/RFP imaging) was used for imaging. Videos of endosomes were taken for 1 min, with pictures in 1-s intervals.

Protein extraction was performed as previously described.^{38 (link)} Protein lysates were separated by SDS–PAGE and blotted onto PVDF membranes. Western blot analysis was performed according to standard techniques. Antibodies used were as follows: α-actin (Abcam, Cambridge, UK; ab8227), α-LC3B (Cell Signaling, Danvers, MA, USA; #2775), α-Myc-Tag (Millipore, Darmstadt, Germany; 05 – 724), α-GFP (Covance, Princeton, NJ, USA; B34), α-p53 DO1 (BD Bioscience, Oxford, UK; #554293), α-p62 (Sigma; p0067), α-BSA (Santa Cruz, Dallas, TX, USA; clone 25G7) and α-COX IV (Abcam; ab16056).