Figure 5.

FIP200 binding site is essential for Atg13 function. (A) atg13^−/− cells were reconstituted with HA-tagged versions of splice variants A-G and incubated in full medium (RPMI) or EBSS for 1 h in the presence or absence of 10 nM BafA1. Equal protein amounts from cleared cellular lysates were analyzed for Atg13, HSP90 and LC3 by immunoblotting. LC3-II/HSP90 ratios from three independent experiments are represented as mean values ± SEM (B) atg13^−/− retrovirally transfected with cDNAs encoding either HA-Atg13 isoform A (full-length) or HA-Atg13 isoform C (?exon12) or the empty vector were stably transfected with pmRFP-EGFP-rLC3. Cells were incubated in EBSS for 2 h and analyzed by confocal laser scanning microscopy. The mRFP signal is displayed in red and the EGFP signal in green in the merged image. The percentage of autolysosome containing cells (>100 cells/experiment) from three independent experiments is represented as mean value ± SD *p < 0.05, Student’s t-test.