Journal: The Journal of biological chemistry

Article Title: Identification of quinone-binding and heme-ligating residues of the smallest membrane-anchoring subunit (QPs3) of bovine heart mitochondrial succinate:ubiquinone reductase.

doi: 10.1074/jbc.274.13.8717

Figure Lengend Snippet: FIG. 1. Identification of recombinant QPs3 by Western blot. A, SDS-PAGE of succinate:ubiquinone reductase (lane 2), recombinant GST-QPs3 fusion protein (lane 3), thrombin-treated fusion protein (lane 4), purified recombinant QPs3 (lane 5). The molecular mass standard containing phosphorylase B (108 kDa), bovine serum albumin (84 kDa), ovalbumin (53 kDa), carbonic anhydrase (35 kDa), soybean trypsin inhibitor (28 kDa), and lysozyme (20 kDa) is in the lane 1. The proteins in A were electrophoretically transferred to nitrocellulose membrane and reacted with anti-QPs3 peptide antibodies (B) and anti-GST-QPs3 antibodies (C). Protein A horseradish peroxidase conjugate was used as a second antibody.

Article Snippet: Agarose, acrylamide, bisacrylamide, horseradish peroxidase color development reagent, protein A horseradish peroxidase conjugate, and Bradford reagent were obtained from Bio-Rad.

Techniques: Recombinant, Western Blot, SDS Page, Purification, Membrane

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