PLEKHM1 binds Arl8b to mediate autophagosome–lysosome fusion. (a) U2OS cells were transfected with vector alone (control), FLAG-PLEKHM1 (WT), -PLEKHM1 (HRR→A), or -NΔ300 PLEKHM1 constructs and subjected to 2 h of starvation using EBSS media in the presence or absence of Baf A1. Lysates from these cell types were immunoblotted (IB) with the indicated antibodies. (b–d) Representative confocal micrographs of HeLa cells expressing ptf-LC3B alone, cotransfected with FLAG-PLEKHM1 (WT) or FLAG-PLEKHM1 (HRR→A), and starved for 2 h in EBSS. Red-only punctate structures in magnified insets represent autolysosomes marked by white arrowheads, and yellow punctate structures represent autophagosomes marked by yellow arrowheads. (e) Yeast two-hybrid assay. Cotransformants were spotted on -Leu-Trp and -Leu-Trp-His media to confirm viability and interactions, respectively. (f) U2OS cells treated with the indicated siRNAs were given Baf A1 treatment in normal growth medium. Lysates were immunoblotted with the indicated antibodies. The levels of LC3B-II normalized to α-tubulin were quantified using densitometric analysis as shown. (g) U2OS cells treated with indicated siRNAs were further subjected to the following treatments: normal growth medium or starvation in EBSS media for 2 h with or without Baf A1. The lysates were immunoblotted for the indicated antibodies. Bars: (main) 10 µm; (insets) 2 µm.