Figure 6.

Overexpression of the SH3 domain of Bif-1 but not of Endophilin A1 suppresses autophagosome formation. (a) HeLa cells were transfected with 0.2 μg of GFP-LC3 and 0, 0.2 or 0.6 μg of Bif-1(SH3)-Myc expression plasmids for 24 h. The total amount of plasmid DNA was adjusted at 0.8 μg per transfection with the pcDNA3 vector. The cells were then cultured in EBSS or CM for 3 h and the number of GFP-LC3 dots per GFP-positive cell was determined using a fluorescent microscope (mean ± s.d.; n = 40). (b) HeLa cells were transfected with GFP-LC3 in combination with either Bif-1 (SH3)-HcRed, Endophilin A1 (SH3)-HcRed or empty HcRed vector for 20 h. The cells were then cultured in EBSS or CM for 2 h and the number of GFP-LC3 dots per HcRed-positive cell was counted using a fluorescent microscope (mean ± s.d.; n = 46). Statistical significance in a and b was determined by Student's t test (* p < 0.0001). (c) 293T cells were transfected with the indicated plasmids for 24 h and subjected to immunoprecipitation with anti-Flag monoclonal antibody. The resulting immune complexes and TCL were analyzed by immunoblotting with anti-Myc and anti-Flag polyclonal antibodies. The full scans of the immunoblots shown in c are presented in the Supplementary Information, Fig. S5.