Journal: PLoS ONE

Article Title: Human FGF-21 Is a Substrate of Fibroblast Activation Protein

doi: 10.1371/journal.pone.0151269

Figure Lengend Snippet: (A) Human FGF-21 is cleaved by FAP. Recombinant human FGF-21 was digested by recombinant human FAP and visualized by Coomassie staining of SDS-Page gel. (B) Time course of FGF-21 digestion by FAP quantified by LC/MS extracted ion integration of peaks corresponding to intact (1–181) and cleaved (1–171) forms of FGF-21 (n = 3 per time point per group). Values are mean ± SEM with one phase decay curve fit superimposed. (C) FAP cleavage of FGF-21 is prevented by ARI-3099. ARI-3099 was pre-incubated with recombinant FAP for 30 minutes prior to addition of FGF-21. Reaction products were visualized by Coomassie staining of SDS-Page gel. (D) Recombinant PREP does not cleave FGF-21. Recombinant human PREP was added to recombinant FGF-21 and visualized by Coomassie staining of SDS-Page gel.

Article Snippet: Reactions were carried out at a final concentration of 20 μM FGF-21, 200 nM recombinant human FAP (R&D systems) or PREP (R&D systems) and 16 μM ARI-3099.

Techniques: Recombinant, Staining, SDS Page, Liquid Chromatography with Mass Spectroscopy, Incubation

Journal: PLoS ONE

Article Title: Human FGF-21 Is a Substrate of Fibroblast Activation Protein

doi: 10.1371/journal.pone.0151269

Figure Lengend Snippet: (A) FAP cleaves human FGF-21 in mouse, monkey and human plasma. Recombinant FGF-21 was added to plasma to a final concentration of 1 μM in the presence or absence of 16 μM ARI-3099 followed by assessment of intact FGF-21 by sandwich ELISA (n = 3 per group). Values are mean ± SEM. *P < .05 ***P < .001 by ANOVA . (B) FAP activity of mouse, monkey and human plasma as assessed by the FAP-specific fluorescent substrate ARI-3144.

Article Snippet: Reactions were carried out at a final concentration of 20 μM FGF-21, 200 nM recombinant human FAP (R&D systems) or PREP (R&D systems) and 16 μM ARI-3099.

Techniques: Clinical Proteomics, Recombinant, Concentration Assay, Sandwich ELISA, Activity Assay

Journal: Cell Death & Disease

Article Title: Direct conversion of human umbilical cord mesenchymal stem cells into retinal pigment epithelial cells for treatment of retinal degeneration

doi: 10.1038/s41419-022-05199-5

Figure Lengend Snippet: The expression level of PTPN13 in shCont-iRPE and shPtpn13-iRPE cells were detected by A Western blotting and B quantitative analysis ( n = 3, P value measured by Student’s unpaired t test). C Representative images of shCont-iRPE and shPtpn13-iRPE cells. RPE-specific and EMT-associated markers in shCont-iRPE cells and shPtpn13-iRPE cells stimulated with TGF-β1 or TGF-β2 were detected by D immunostaining, E Western blotting, and F quantitative analysis ( n = 3, P value measured by one-way ANOVA and post hoc Bonferroni’s test). Phosphorylation of SMAD2/3, p38, and ERK1/2 in shCont-iRPE and shPtpn13-iRPE cells were analyzed by G immunostaining, H Western blotting, and I quantitative analysis ( n = 3, P value measured by one-way ANOVA and post hoc Bonferroni’s test). Scale bar = 50 μm. Results are expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, shPtpn13-TGF-β1 compared with shCont-TGF-β1; # P < 0.01, ## P < 0.01, ### P < 0.001, shPtpn13-TGF-β2 compared with shCont-TGF-β2.

Article Snippet: After blocked with 3% BSA in PBS for 1 h, membranes were incubated with primary antibodies against RPE65 (1:1000, Novus Biologicals, Centennial, CO, USA); against TYRP1 (1:1000), MERTK (1:1000), FN1 (1:1000), α-SMA (1:2000), pSMAD2 (1:500), SMAD2 (1:1000), pSMAD3 (1:1000), SMAD3 (1:1000) TGF-β receptor1 (TGF-βR1) (1:1000), Abcam; against pERK1/2 (1:1000), ERK1/2 (1:1000), CST; against Claudin19 (1:1000, Invitrogen); against Flag (1:2000, MBL International, Woburn, MD, USA); against CRALBP (1:1000), syntenin1 (1:1000), DUSP4 (1:1000), DUSP10 (1:1000), PHLPP1 (1:1000), PPM1L (1:1000), PPP1CC (1:1000), PPP2R5A (1:1000), PTPN13 (1:1000), TGF-βR2 (1:1000), and β-Actin (1:5000), Proteintech; for 12 h at 4 °C, followed by incubation with corresponding secondary antibodies for 1 h at room temperature.

Techniques: Expressing, Western Blot, Immunostaining, Phospho-proteomics

Journal: Cell Death & Disease

Article Title: Direct conversion of human umbilical cord mesenchymal stem cells into retinal pigment epithelial cells for treatment of retinal degeneration

doi: 10.1038/s41419-022-05199-5

Figure Lengend Snippet: PTPN13 binding syntenin1 in iRPE cells were identified by A mass spectrometry and confirmed by B , C Western blotting. Phosphorylation of syntenin1 was detected by D anti-phosphorylated-Tyr antibody CO-IP assay and E quantitative analysis ( n = 3). The expression levels of TGF-βR1 and TGF-βR2 in shCont-iRPE and shPtpn13-iRPE cells were detected by F Western blotting, G quantitative analysis ( n = 3). The expression levels of TGF-βR1 and TGF-βR2 on cell membranes of shCont-iRPE and shPtpn13-iRPE cells were detected by H flow cytometry and I quantitative analysis of mean fluorescence density ( n = 3). Results are expressed as mean ± SD. P value measured by Student’s unpaired t test.

Article Snippet: After blocked with 3% BSA in PBS for 1 h, membranes were incubated with primary antibodies against RPE65 (1:1000, Novus Biologicals, Centennial, CO, USA); against TYRP1 (1:1000), MERTK (1:1000), FN1 (1:1000), α-SMA (1:2000), pSMAD2 (1:500), SMAD2 (1:1000), pSMAD3 (1:1000), SMAD3 (1:1000) TGF-β receptor1 (TGF-βR1) (1:1000), Abcam; against pERK1/2 (1:1000), ERK1/2 (1:1000), CST; against Claudin19 (1:1000, Invitrogen); against Flag (1:2000, MBL International, Woburn, MD, USA); against CRALBP (1:1000), syntenin1 (1:1000), DUSP4 (1:1000), DUSP10 (1:1000), PHLPP1 (1:1000), PPM1L (1:1000), PPP1CC (1:1000), PPP2R5A (1:1000), PTPN13 (1:1000), TGF-βR2 (1:1000), and β-Actin (1:5000), Proteintech; for 12 h at 4 °C, followed by incubation with corresponding secondary antibodies for 1 h at room temperature.

Techniques: Binding Assay, Mass Spectrometry, Western Blot, Phospho-proteomics, Co-Immunoprecipitation Assay, Expressing, Flow Cytometry, Fluorescence

Journal: Cell Death & Disease

Article Title: Direct conversion of human umbilical cord mesenchymal stem cells into retinal pigment epithelial cells for treatment of retinal degeneration

doi: 10.1038/s41419-022-05199-5

Figure Lengend Snippet: Knockdown efficiency of syntenin1 was determined by A qRT-PCR ( n = 3, P value measured by one-way ANOVA and post hoc Bonferroni’s test), B Western blotting, and C quantitative analysis ( n = 3, P value measured by one-way ANOVA and post hoc Bonferroni’s test). RPE-specific and EMT-associated markers in shCont-shPtpn13-iRPE and shSyntenin1-shPtpn13-iRPE cells were analyzed by D Western blotting and E quantitative analysis ( n = 3, P value measured by one-way ANOVA and post hoc Bonferroni’s test). Phosphorylation of SMAD2/3, p38, and ERK1/2 in shCont-shPtpn13-iRPE and shSyntenin1-shPtpn13-iRPE cells were analyzed by F Western blotting and G quantitative analysis ( n = 3, P value measured by one-way ANOVA and post hoc Bonferroni’s test). The expression levels of TGF-βR1 and TGF-βR2 on the cell membranes of shCont-shPtpn13-iRPE and shSyntenin1-shPtpn13-iRPE cells were detected by H flow cytometry and I quantitative analysis of mean fluorescence density ( n = 3, P value measured by Student’s unpaired t test). J Schematic model for the PTPN13 dephosphorylating syntenin1 mediates TGF-β receptor internalization and degradation. Results are expressed as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, shCont-shPtpn13-TGF-β1 compared with shSyntenin1-shPtpn13-TGF-β1; # P < 0.01, ## P < 0.01, ### P < 0.001, shCont-shPtpn13-TGF -β 2 compared with shSyntenin1-shPtpn13-TGF-β2.

Article Snippet: After blocked with 3% BSA in PBS for 1 h, membranes were incubated with primary antibodies against RPE65 (1:1000, Novus Biologicals, Centennial, CO, USA); against TYRP1 (1:1000), MERTK (1:1000), FN1 (1:1000), α-SMA (1:2000), pSMAD2 (1:500), SMAD2 (1:1000), pSMAD3 (1:1000), SMAD3 (1:1000) TGF-β receptor1 (TGF-βR1) (1:1000), Abcam; against pERK1/2 (1:1000), ERK1/2 (1:1000), CST; against Claudin19 (1:1000, Invitrogen); against Flag (1:2000, MBL International, Woburn, MD, USA); against CRALBP (1:1000), syntenin1 (1:1000), DUSP4 (1:1000), DUSP10 (1:1000), PHLPP1 (1:1000), PPM1L (1:1000), PPP1CC (1:1000), PPP2R5A (1:1000), PTPN13 (1:1000), TGF-βR2 (1:1000), and β-Actin (1:5000), Proteintech; for 12 h at 4 °C, followed by incubation with corresponding secondary antibodies for 1 h at room temperature.

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Phospho-proteomics, Expressing, Flow Cytometry, Fluorescence