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. 2008 Oct 20;29(1):157–171. doi: 10.1128/MCB.01082-08

FIG. 6.

FIG. 6.

Membrane targeting signal within the CTDs of ULK1 and ULK2. (A) 293/GFP-LC3 cells were transfected with Myc-ULK1 and starved in EBSS-leupeptin for 2 h. Cell homogenates were centrifuged at 100,000 × g to isolate membrane (Memb) and supernatant (Sup) fractions. Aliquots of the supernatant (representing 2% of the cell sample) and membrane (representing 10% of the cell sample) fractions were analyzed by SDS-PAGE. The top half of the blot was probed with anti-Myc and the bottom with anti-LC3. Where indicated, the membrane fraction was washed with HB containing 0.15 M or 0.5 M KCl. (B and C) Myc-tagged ULK1 and -2 constructs were transfected into cells and analyzed for membrane association as described above. (D) After transfection with Myc-tagged ULK1 CTD, cells were left untreated or starved in EBSS-leupeptin for 2 h. Where indicated, the membrane pellet was washed in HB containing 0.5 M KCl and analyzed as described above. (E) 293/GFP-LC3 cells were transfected with Myc-ULK1 CTD for 24 h and then starved in EBSS-leupeptin for 2 h before fixation and immunostaining with anti-Myc monoclonal antibody. In a Z section close to the substratum (Z = 0), ULK1 CTD strongly inhibited GFP-LC3 punctum formation. In the Z + 1 section, Myc-ULK1 CTD could be detected colocalizing with GFP-LC3-positive structures (inset). Bar = 10 μm. (F) Membrane associations of ULK1 CTD (Full CTD) and various C-terminal deletion constructs in starved 293/GFP-LC3 cells. See the schematic in Fig. 2 for the positions of inserted stop codons. The expression of ULK1 CTD was detected with anti-Myc, and GFP-LC3 was detected with anti-LC3. (G) Membrane associations of ULK1 CTD and two N-terminal deletion constructs, Myc-LHS (845-1051) and Myc-LKG (864-1051), in starved 293/GFP-LC3 cells. (H) 293/GFP-LC3 cells were transfected with full-length Myc-ULK1 or -ULK2 and then left untreated or starved in EBSS-leupeptin for 2 h before isolation of supernatant and membrane fractions. Expressed proteins were detected as described above. (I) Membrane association of transfected Myc-ULK1 in starved 293/GFP-LC3 cells. Membrane pellets were analyzed before washes (none) or following extraction in HB (0) or HB supplemented with 1.0% TX-100. (J) Membrane association of endogenous ULK1 was analyzed in untransfected, starved 293/GFP-LC3 cells following the fractionation and wash procedures described for panel I. WT, wild type; Untrans, untransfected.