Fig. 5

a A schematic representation of M6P modification and sorting of lysosomal enzymes. b CTSD protein level in the whole cell lysate and conditioned media of HEK293T WT, TMEM251 KO (251-KO), GNPTAB KO (G-KO), CI-MPR KO (CI-KO) cells (n = 3 independent replicates). Asterisk: a non-specific band. c CI-MPR binding assay of conditioned media from TMEM251 KO, GNPTAB KO, CI-MPR KO, and CI-MPR and TMEM251 double KO cells (n = 3 independent replicates). d–f Detection of M6P modification of LIPA (d), CTSD (e), and CTSZ (f) in HEK293T and sgTMEM251 cells (n = 2 independent replicates) using single-chain antibodies against M6P (scFv M6P). Asterisk: a non-specific band. g, h Rescue of TMEM251 KO with conditioned media from GNPTAB KO and CI-MPR KO cells. i–l Quantification of the full-length LAPTM4A, LC3B-II, mature CTSC, and mature CTSD protein levels in h. Mean of 3 independent replicates is shown. Error bars represent standard deviation. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. See source data file for exact P values.