Posttranslational regulation of ULK1 levels in erythroid cells and splenocytes from mtDNA-mutator mice. Representative immunoblots (A) and graphs (B) showing the steady-state levels of p4EBP1, total 4EBP1, and GAPDH in young (2-month-old) and old (8- to 12-month-old) mice. The ratio of phosphorylation to total 4EBP1 was normalized to that of wild-type animals for each set of mice. Representative immunoblots (C) and graphs (D) showing the steady-state levels of ribosomal proteins RPL5 and RPS19 in spleens of young and old mice. n = 2 mice per genotype (young) or 3 mice per genotype (old). (E-G) Representative immunoblots and graphs (mean ± standard deviation) showing steady-state levels of P62 (E-F) and ULK1 (E,G) in spleens of young and old mice. n = 2 mice per genotype per time point. (H-I) Lin⁻/Ter119⁺ erythroid cells were selected from bone marrow of phlebotomized mice by using magnetic beads. Extracts prepared from sorted populations were subjected to immunoblot analyses using antibodies against ULK1 and GAPDH. The wild-type and Polg^mt/mt lanes in the representative immunoblots (H) are separated by white lines to indicate juxtaposition of 2 nonadjacent from the same gel. The graph (mean ± SEM) shows reduced ULK1 levels (normalized to GAPDH) in erythroid cells of mtDNA-mutator mice. n = 3 (wild-type) or 4 (PolgA^mt/mt) mice. P < .01. (J) Splenocytes were isolated from spleens of old mtDNA-mutator mice and incubated with or without the indicated drugs for 3 hours: 250 nM Torin1, 25 nM rapamycin (Rap), or 25 nM MG132. ULK1 immunoprecipitates were analyzed by immunoblot analyses for ULK1 phosphorylated at serine 757 (by using 4/5 of the immunoprecipitate) and total ULK1 (by using 1/5 of the immunoprecipitate). The experiment was performed 3 times; a representative experiment is shown.