Journal: Nature Communications

Article Title: GCAF(TMEM251) regulates lysosome biogenesis by activating the mannose-6-phosphate pathway

doi: 10.1038/s41467-022-33025-1

Figure Lengend Snippet: a CHX chase assay of stably expressed GFP-RNF152 in sgRNA control, sgRNA-1 TMEM251, and sgRNA-2 TMEM251 cells. b Steady-state (0 h) protein levels in a . Mean of 3 independent replicates is shown. Error bars represent standard deviation. ** p ≤ 0.01, *** p ≤ 0.001. c Quantification of GFP-RNF152 degradation in a . Mean of 3 independent replicates is shown. Error bars represent standard deviation. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Table: Calculated protein half-lives. d CHX chase assay of endogenous LAPTM4A in sgRNA control, sgRNA-1 TMEM251, and sgRNA-2 TMEM251 cells. Arrowhead: cleavage product of LAPTM4A. e Steady-state (0 h) LAPTM4A protein levels in d . Mean of 3 independent replicates is shown. Error bars represent standard deviation. ** p ≤ 0.01, *** p ≤ 0.001. f Quantification of LAPTM4A degradation in d , Mean of 3 independent replicates is shown. Error bars represent standard deviation. ** p ≤ 0.01. Table: Calculated protein half-lives. g EGFR degradation assay in HeLa WT and TMEM251 KO cells. h Quantification of EGFR degradation in g . Mean of 3 independent replicates is shown. Error bars represent standard deviation. * p ≤ 0.05. Table: Calculated protein half-lives. i p62 and LC3B protein levels in HEK293 WT and sgTMEM251 cells. j , k Quantification of the p62 ( j ) and LC3B-II ( k ) protein levels in ( i ). Mean of 3 independent replicates is shown. Error bars represent standard deviation. * p ≤ 0.05. ** p ≤ 0.01. See source data file for exact P values.

Article Snippet: To detect TMEM251, M6P (scFv M6P), or GNPTAB-3xHA KI, the anti-protein A HRP (PA00-03, Rockland, for TMEM251 and M6P) or mouse HRP (115-035-046, Jackson labs, for GNPTAB-3xHA KI) secondary antibodies were used at 1:10,000 dilution.

Techniques: Stable Transfection, Control, Standard Deviation, Degradation Assay

Journal: Nature Communications

Article Title: GCAF(TMEM251) regulates lysosome biogenesis by activating the mannose-6-phosphate pathway

doi: 10.1038/s41467-022-33025-1

Figure Lengend Snippet: 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.

Article Snippet: To detect TMEM251, M6P (scFv M6P), or GNPTAB-3xHA KI, the anti-protein A HRP (PA00-03, Rockland, for TMEM251 and M6P) or mouse HRP (115-035-046, Jackson labs, for GNPTAB-3xHA KI) secondary antibodies were used at 1:10,000 dilution.

Techniques: Modification, Binding Assay, Standard Deviation

Journal: Nature Communications

Article Title: GCAF(TMEM251) regulates lysosome biogenesis by activating the mannose-6-phosphate pathway

doi: 10.1038/s41467-022-33025-1

Figure Lengend Snippet: a A schematic representation of GNPTAB processing by S1P. b The processing of the endogenously tagged GNPTAB in TMEM251 KO and TMEM251 overexpression (OE) cells. c Quantification of the GNPTAB processing efficiency in b . Mean of 3 independent replicates is shown. Error bars represent standard deviation. **** p ≤ 0.0001. d A schematic representation of SREBP2 processing by S1P and S2P. e The processing of SREBP2 in HEK293T WT and TMEM251 KO cells. f Quantification of the SREBP2 processing efficiency in e . Mean of 3 independent replicates is shown. Error bars represent standard deviation. ** p ≤ 0.01, *** p ≤ 0.001. g A schematic representation of ATF6 processing by S1P and S2P. h ATF6 processing in HEK293T WT and TMEM251 KO cells after 1 h of CHX and DTT treatment. i Quantification of the ATF6 processing efficiency in ( h ). Mean of 3 independent replicates is shown. Error bars represent standard deviation. ** p ≤ 0.01. See source data file for exact P values. j , k Reciprocal IP ( n = 2 independent replicates) showing interactions between GNPTAB and TMEM251. l , m Reciprocal IP ( n = 2 independent replicates) showing interactions between S1P (S414A) and TMEM251.

Article Snippet: To detect TMEM251, M6P (scFv M6P), or GNPTAB-3xHA KI, the anti-protein A HRP (PA00-03, Rockland, for TMEM251 and M6P) or mouse HRP (115-035-046, Jackson labs, for GNPTAB-3xHA KI) secondary antibodies were used at 1:10,000 dilution.

Techniques: Over Expression, Standard Deviation

Journal: Nature Communications

Article Title: GCAF(TMEM251) regulates lysosome biogenesis by activating the mannose-6-phosphate pathway

doi: 10.1038/s41467-022-33025-1

Figure Lengend Snippet: TMEM251/GCAF deficiency leads to defects in M6P modification of lysosomal enzymes at the cis-Golgi. Lysosomal enzymes without M6P are targeted to the secretory pathway, resulting in lysosome dysfunction.

Article Snippet: To detect TMEM251, M6P (scFv M6P), or GNPTAB-3xHA KI, the anti-protein A HRP (PA00-03, Rockland, for TMEM251 and M6P) or mouse HRP (115-035-046, Jackson labs, for GNPTAB-3xHA KI) secondary antibodies were used at 1:10,000 dilution.

Techniques: Modification