Generation of viable ulk1^−/− mice without significant defect in autophagy. (A) The first panel shows the genomic organization of the 5′ end of the ulk1 gene. The second panel depicts the targeting construct that introduces an ulk1 allele where loxP sites flank a 5.6-kb EcoRI fragment containing exons I to III. The third panel shows the expected sizes of the wild-type (WT) and floxed (FL) ulk1 alleles, following digestion of DNA with BamHI after probing with the 1-kb EcoRI-BamHI probe (indicated by the bar). (B) Southern blot analysis of BamHI digested DNA from representative wild-type (WT) and floxed (FL) ulk1 clones probed with the EcoRI-BamHI probe. This probe generates an approximately 19-kb fragment in WT mice, but as a result of cloning into to the loxP vector a BamHI restriction site is introduced that produces a 4.3-kb fragment in clones containing a homologously recombined floxed ulk1 allele. (C) Northern blot analysis of ulk1^+/+ (WT) and ulk1^−/− (KO) murine embryonic fibroblasts (MEFs). (D) Western blot analysis of ulk1^+/+ (WT) and ulk1^−/− (KO) MEFs. The asterisk (*) denotes nonspecific bands. (E) Western blot analysis of ulk1^+/+ (WT) and ulk1^−/− (KO) MEFs cultured in the presence (C) or absence of glucose (−gluc) for 24 or 48 hours. The experiment was performed in triplicate. Error bars represent standard deviation. Percentage LC3 conversion was calculated using the following formula: 100*LC3-I/total LC3.