Journal: bioRxiv

Article Title: High efficiency organoid-derived cyst cultures as a drug discovery platform for polycystic kidney disease

doi: 10.1101/2025.02.16.638545

Figure Lengend Snippet: A . Schematic of the protocol. B . Day (d) 12 nephron organoids differentiated from wildtype iPSCs. C . Enzymatic digestion of whole nephron organoids into a single cell suspension, marking d0 of UB differentiation. D . Development of UB organoids over 15 days. E . qPCR analysis comparing d12 nephron organoids and d7 UB organoids for nephron and UB marker gene expression. ** p≤ 0.01; *** p≤ 0.001; **** p≤ 0.0001 (Unpaired t -test). F . Immunohistological characterization of UB organoids showing expression of distal tubule (CDH1, PAX2, GATA3) and UB markers (RET, KRT8). Nuclear counterstain, Hoechst. Scale bars: 400 µm (B-D); 100 µm (F).

Article Snippet: Antibodies used were: PAX2 (BioLegend 901001), CDH1 (BD Biosciences 610181), RET (Novus NBP184568), GATA3 (Thermo MA1-028), KRT8 (DSHB TROMA), EPCAM (Sigma HPA026761).

Techniques: Suspension, Marker, Gene Expression, Expressing

Journal: bioRxiv

Article Title: High efficiency organoid-derived cyst cultures as a drug discovery platform for polycystic kidney disease

doi: 10.1101/2025.02.16.638545

Figure Lengend Snippet: A . Development of PKD2 -/- UB organoids over 25 days. Arrow shows an early cyst on d7. B . Quantification of cyst development in wildtype and PKD2 -/- UB organoids. C . PKD2 -/- cyst diameters increase over time. D . Example of a high efficiency cyst culture at d17. E . Upregulation of ADPKD marker genes in PKD2 -/- compared to wildtype UB organoids. ** p≤ 0.01; *** p≤ 0.001; **** p≤ 0.0001 (Unpaired t -test). F . Immunohistological analysis of PKD2 -/- UB organoids, showing expression of distal tubule and UB markers CDH1, PAX2, RET and GATA3 in the cystic epithelium. Nuclear counterstain, Hoechst. Scale bars: 400 µm (A); 1000 µm (D); 100 µm (F).

Article Snippet: Antibodies used were: PAX2 (BioLegend 901001), CDH1 (BD Biosciences 610181), RET (Novus NBP184568), GATA3 (Thermo MA1-028), KRT8 (DSHB TROMA), EPCAM (Sigma HPA026761).

Techniques: Marker, Expressing

Journal: Cell Death Discovery

Article Title: TRIM27-controlled endothelium-derived exosomes play a central role in podocyte injury in diabetic kidney disease

doi: 10.1038/s41420-026-02953-y

Figure Lengend Snippet: A Double immunofluorescence staining of TRIM27 protein in glomerular endothelial cells of DKD patients. Kidney sections were costained for TRIM27 (green) and specific endothelial cell marker CD31 (red). Scale bars: 10 μm. B Significant positive correlation was observed between TRIM27 expression and proteinuria in DKD patients ( n = 36 DKD patients). C , D The ELISA assays showed serum contents of syndecan-1 and VCAM-1 increased in DKD patients. *** P < 0.001 vs. Normal group ( n = 24 normal controls and 36 DKD patients). E , F Significant positive correlation was observed between TRIM27 expression and syndecan-1 and VCAM-1 serum levels in DKD patients ( n = 36 DKD patients). G Expression of TRIM27 (green) in glomerular endothelial cells (specific marker, CD31; red) of STZ-induced diabetec mice detected by immunofluorescence. Scale bars: 10 μm. H Expression of syndecan-1 in the glomerulus of STZ mice detected by immunofluorescence. Scale bars: 25 μm. I Significant negative correlation was observed between TRIM27 and syndecan-1 expression in STZ mice ( n = 6 mice, 10 glomeruli per mouse). J – L Western blot assay showed TRIM27 expression increased in HRGECs treated with HG or TGF-β1. ** P < 0.01, **** P < 0.0001 vs. 0 h group ( n = 3). M IF assay showed that the expression of TRIM27 increased in HRGECs treated with TGF-β1. Scale bars: 25 μm. N – P Western blot assay showed VCAM-1 expression increased in HRGECs treated with HG or TGF-β1. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. 0 h group ( n = 3). Q , R The ELISA assays showed the contents of syndecan-1 and VCAM-1 increased in cultures supernatant of HRGECs treated with HG for 24 h. ** P < 0.01, **** P < 0.0001 vs. 0 h group ( n = 3). S NO level increased in the HRGECs after treated with HG. ** P < 0.01, **** P < 0.0001 vs. 0 h group ( n = 3). Student’s t test and Bonferroni’s correction were performed to analyze statistical significance. Pearson’s method was used for correlation analysis. Data are the mean ± SEM.

Article Snippet: Then, the membranes were incubated overnight at 4 °C with 1:1000 dilutions of antibodies against TRIM27 (Proteintech, #12205-1-AP), VCAM-1 (Abcam, #ab134047, RRID: AB_2721053), Janus kinase 2 (JAK2; Proteintech, #17670-1-AP, RRID: AB_2811138), p-JAK2 (Y1007) (Abcam, #ab195055), signal transducer and activator of transcription 3 (STAT3; Proteintech, #60199-1-Ig, RRID: AB_10913811), p-STAT3 (Tyr705) (CST, #9145, RRID: AB_2491009), nephrin (Abcam, #ab58968, RRID: AB_944400), podocin (Abcam, #ab50339, RRID: AB_882097), CD63 (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-5275, RRID: AB_627877), CD9 (Santa Cruz Biotechnology, #sc-13118, RRID: AB_627213), TSG101 (Proteintech, #28283-1-AP, RRID: AB_2881104), Calnexin (Proteintech, #10427-2-AP, RRID: AB_2069033), Rab27a (Proteintech, #17817-1-AP, RRID: AB_2176728), PTEN (Abcam, #ab32199, RRID: AB_777535), Akt (Proteintech, #60203-2-Ig, RRID: AB_10912803), and p-Akt (S473) (Abcam, #ab81283, RRID: AB_2224551).

Techniques: Double Immunofluorescence Staining, Marker, Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Western Blot

Journal: Cell Death Discovery

Article Title: TRIM27-controlled endothelium-derived exosomes play a central role in podocyte injury in diabetic kidney disease

doi: 10.1038/s41420-026-02953-y

Figure Lengend Snippet: A – F Western blot assay showed TRIM27 and VCAM-1 expression decreased in HRGECs treated with shTRIM27 and HG or TGF-β1 for 24 h. * P < 0.05, *** P < 0.001, **** P < 0.0001 vs. control group, # P < 0.05, #### P < 0.0001 vs. HG+shNC group, ## P < 0.01, ### P < 0.001 vs. TGF-β1+shNC group ( n = 3). G , H ELISA assays showed contents of syndecan-1 and VCAM-1 decreased in culture supernatants after TRIM27 knockdown in HRGECs and treatment with HG for 24 h. * P < 0.05, **** P < 0.0001 vs. control group, ## P < 0.01, #### P < 0.0001 vs. HG+shNC group ( n = 3). I – K IF assay showed that downregulation of TRIM27 increased VE-cadherin, ZO-1, and syndecan-1 expression in HRGECs treated with TGF-β1 for 24 h. Scale bars: 25 μm. Bonferroni’s correction was performed to analyze statistical significance. Data are the mean ± SEM.

Article Snippet: Then, the membranes were incubated overnight at 4 °C with 1:1000 dilutions of antibodies against TRIM27 (Proteintech, #12205-1-AP), VCAM-1 (Abcam, #ab134047, RRID: AB_2721053), Janus kinase 2 (JAK2; Proteintech, #17670-1-AP, RRID: AB_2811138), p-JAK2 (Y1007) (Abcam, #ab195055), signal transducer and activator of transcription 3 (STAT3; Proteintech, #60199-1-Ig, RRID: AB_10913811), p-STAT3 (Tyr705) (CST, #9145, RRID: AB_2491009), nephrin (Abcam, #ab58968, RRID: AB_944400), podocin (Abcam, #ab50339, RRID: AB_882097), CD63 (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-5275, RRID: AB_627877), CD9 (Santa Cruz Biotechnology, #sc-13118, RRID: AB_627213), TSG101 (Proteintech, #28283-1-AP, RRID: AB_2881104), Calnexin (Proteintech, #10427-2-AP, RRID: AB_2069033), Rab27a (Proteintech, #17817-1-AP, RRID: AB_2176728), PTEN (Abcam, #ab32199, RRID: AB_777535), Akt (Proteintech, #60203-2-Ig, RRID: AB_10912803), and p-Akt (S473) (Abcam, #ab81283, RRID: AB_2224551).

Techniques: Western Blot, Expressing, Control, Enzyme-linked Immunosorbent Assay, Knockdown

Journal: Cell Death Discovery

Article Title: TRIM27-controlled endothelium-derived exosomes play a central role in podocyte injury in diabetic kidney disease

doi: 10.1038/s41420-026-02953-y

Figure Lengend Snippet: A – D Western blot assay showed p-JAK2 (Y1007) and p-STAT3 (Tyr705) expression increased in HRGECs treated with HG or TGF-β1. ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. 0 h group ( n = 3). E , F Western blot assay showed p-JAK2 (Y1007), p-STAT3 (Tyr705), and VCAM-1 expression decreased after inhibition of the JAK2/STAT3 pathway by AG490 in HRGECs and treatment with HG for 24 h. ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. control group, ## P < 0.01 vs. HG + DMSO group ( n = 3). G – I IF assay showed that VE-cadherin, ZO-1, and syndecan-1 expression increased in HRGECs treated with AG490 and TGF-β1 for 24 h. Scale bars: 25 μm. J , K The ELISA assays showed contents of syndecan-1 and VCAM-1 in culture supernatants of HRGECs decreased after treated with AG490 and HG for 24 h. * P < 0.05, **** P < 0.0001 vs. control group, # P < 0.05, ### P < 0.001 vs. HG + DMSO group ( n = 3). L , M Western blot showed that VCAM-1 expression decreased after inhibition of the JAK2/STAT3 pathway in HRGECs. ** P < 0.01 vs. control group, ### P < 0.001 vs. TGF-β1 + DMSO group ( n = 3). N , O Western blot assay showed downregulation of TRIM27 decreased p-JAK2 (Y1007) and p-STAT3 (Tyr705) expression in HRGECs treated with HG for 2 h. *** P < 0.001, **** P < 0.0001 vs. control group, ### P < 0.001 vs. HG+shNC group ( n = 3). Bonferroni’s correction was performed to analyze statistical significance. Data are the mean ± SEM.

Article Snippet: Then, the membranes were incubated overnight at 4 °C with 1:1000 dilutions of antibodies against TRIM27 (Proteintech, #12205-1-AP), VCAM-1 (Abcam, #ab134047, RRID: AB_2721053), Janus kinase 2 (JAK2; Proteintech, #17670-1-AP, RRID: AB_2811138), p-JAK2 (Y1007) (Abcam, #ab195055), signal transducer and activator of transcription 3 (STAT3; Proteintech, #60199-1-Ig, RRID: AB_10913811), p-STAT3 (Tyr705) (CST, #9145, RRID: AB_2491009), nephrin (Abcam, #ab58968, RRID: AB_944400), podocin (Abcam, #ab50339, RRID: AB_882097), CD63 (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-5275, RRID: AB_627877), CD9 (Santa Cruz Biotechnology, #sc-13118, RRID: AB_627213), TSG101 (Proteintech, #28283-1-AP, RRID: AB_2881104), Calnexin (Proteintech, #10427-2-AP, RRID: AB_2069033), Rab27a (Proteintech, #17817-1-AP, RRID: AB_2176728), PTEN (Abcam, #ab32199, RRID: AB_777535), Akt (Proteintech, #60203-2-Ig, RRID: AB_10912803), and p-Akt (S473) (Abcam, #ab81283, RRID: AB_2224551).

Techniques: Western Blot, Expressing, Inhibition, Control, Enzyme-linked Immunosorbent Assay

Journal: Cell Death Discovery

Article Title: TRIM27-controlled endothelium-derived exosomes play a central role in podocyte injury in diabetic kidney disease

doi: 10.1038/s41420-026-02953-y

Figure Lengend Snippet: A Eighteen 20-week-old mice were randomly divided into three groups: STZ ( n = 6), STZ+shTRIM27 ( n = 6), and STZ+shNC group ( n = 6). Mice in STZ+shTRIM27 and STZ+shNC groups were renally injected with 50 μl of 1 × 10 11 infective units of adeno-associated virus at three sites each in both kidneys. Six control mice and 6 STZ mice were injected with isometric saline. The mice were sacrificed after 4 weeks. B IF staining showed TRIM27 expression decreased in glomerular endothelial cells of STZ+shTRIM27 mice. Scale bars: 10 μm. C – E Level of 24-h proteinuria, BUN, and Scr decreased in STZ+shTRIM27 mice. ** P < 0.01, *** P < 0.001 vs. Control mice, ## P < 0.01 vs. STZ+shNC mice. n = 6 each group. F , G The ELISA assays showed serum contents of syndecan-1 and VCAM-1 decreased in STZ+shTRIM27 mice. * P < 0.05, *** P < 0.001 vs. Control mice, # P < 0.05, ## P < 0.01 vs. STZ+shNC mice. n = 6 each group. H , I IF staining showed syndecan-1 expression increased in STZ+shTRIM27 mice. Scale bars: 25 μm. *** P < 0.001 vs. Control mice, ### P < 0.001 vs. STZ+shNC mice ( n = 6). J Electron microscopy assay showed ultrastructure of the podocytes in the renal cortex of mice. Scale bars: 2 μm. K – M IHC staining showed WT1 and podocin expression increased in STZ+shTRIM27 mice. Scale bars: 25 μm. *** P < 0.001 vs. Control mice, ## P < 0.01 vs. STZ+shNC mice ( n = 6). N IF staining showed synaptopodin expression increased in STZ+shTRIM27 mice. Scale bars: 10 μm. Bonferroni’s correction was performed to analyze statistical significance. Data are the mean ± SEM.

Article Snippet: Then, the membranes were incubated overnight at 4 °C with 1:1000 dilutions of antibodies against TRIM27 (Proteintech, #12205-1-AP), VCAM-1 (Abcam, #ab134047, RRID: AB_2721053), Janus kinase 2 (JAK2; Proteintech, #17670-1-AP, RRID: AB_2811138), p-JAK2 (Y1007) (Abcam, #ab195055), signal transducer and activator of transcription 3 (STAT3; Proteintech, #60199-1-Ig, RRID: AB_10913811), p-STAT3 (Tyr705) (CST, #9145, RRID: AB_2491009), nephrin (Abcam, #ab58968, RRID: AB_944400), podocin (Abcam, #ab50339, RRID: AB_882097), CD63 (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-5275, RRID: AB_627877), CD9 (Santa Cruz Biotechnology, #sc-13118, RRID: AB_627213), TSG101 (Proteintech, #28283-1-AP, RRID: AB_2881104), Calnexin (Proteintech, #10427-2-AP, RRID: AB_2069033), Rab27a (Proteintech, #17817-1-AP, RRID: AB_2176728), PTEN (Abcam, #ab32199, RRID: AB_777535), Akt (Proteintech, #60203-2-Ig, RRID: AB_10912803), and p-Akt (S473) (Abcam, #ab81283, RRID: AB_2224551).

Techniques: Injection, Virus, Control, Saline, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Electron Microscopy, Immunohistochemistry

Journal: Cell Death Discovery

Article Title: TRIM27-controlled endothelium-derived exosomes play a central role in podocyte injury in diabetic kidney disease

doi: 10.1038/s41420-026-02953-y

Figure Lengend Snippet: A , B Concentrations and size distributions of T+shTRIM27-exo and T+shNC-exo determined by NTA. C Quantification of NTA results from three independent experiments. ** P < 0.01 vs. T+shTRIM27 group ( n = 3). D – G Western blot assay showed downregulation of TRIM27 decreased Rab27a expression in HRGECs treated with TGF-β1 or HG for 24 h. * P < 0.05, *** P < 0.001 vs. control group, # P < 0.05, ## P < 0.01 vs. TGF-β1+shNC or HG+shNC group ( n = 3). H – K Western blot assay showed conditional media from HRGECs with knocked down TRIM27 increased nephrin and podocin expression in HPCs. * P < 0.05, *** P < 0.001 vs. CM group, ## P < 0.01, ### P < 0.001 vs. HM+shNC group or TM+shNC group ( n = 3). L Table of the 10 most abundant miRNAs in HG exosomes compared with control exosomes. M – P Detection of miR-486-5p in HRGEC-derived exosomes and HPCs treated with exosomes by qPCR normalized to U6. **** P < 0.0001 vs. C-exo group, ns. no significance ( n = 6). Student’s t test and Bonferroni’s correction were performed to analyze statistical significance. Values are the mean ± SEM.

Article Snippet: Then, the membranes were incubated overnight at 4 °C with 1:1000 dilutions of antibodies against TRIM27 (Proteintech, #12205-1-AP), VCAM-1 (Abcam, #ab134047, RRID: AB_2721053), Janus kinase 2 (JAK2; Proteintech, #17670-1-AP, RRID: AB_2811138), p-JAK2 (Y1007) (Abcam, #ab195055), signal transducer and activator of transcription 3 (STAT3; Proteintech, #60199-1-Ig, RRID: AB_10913811), p-STAT3 (Tyr705) (CST, #9145, RRID: AB_2491009), nephrin (Abcam, #ab58968, RRID: AB_944400), podocin (Abcam, #ab50339, RRID: AB_882097), CD63 (Santa Cruz Biotechnology, Dallas, TX, USA, #sc-5275, RRID: AB_627877), CD9 (Santa Cruz Biotechnology, #sc-13118, RRID: AB_627213), TSG101 (Proteintech, #28283-1-AP, RRID: AB_2881104), Calnexin (Proteintech, #10427-2-AP, RRID: AB_2069033), Rab27a (Proteintech, #17817-1-AP, RRID: AB_2176728), PTEN (Abcam, #ab32199, RRID: AB_777535), Akt (Proteintech, #60203-2-Ig, RRID: AB_10912803), and p-Akt (S473) (Abcam, #ab81283, RRID: AB_2224551).

Techniques: Western Blot, Expressing, Control, Derivative Assay

Journal: Journal of Biological Chemistry

Article Title: Dok-6, a Novel p62 Dok Family Member, Promotes Ret-mediated Neurite Outgrowth

doi: 10.1074/jbc.m403726200

Figure Lengend Snippet: FIG. 5. Dok-6 binds to Ret via the phosphorylated Ret Tyr1062 residue. A, Dok-6 associates with ligand-activated Ret. Neuro2A cells stably transfected with an expression vector for GFR1 (N2A-1) were deprived of serum for 2 h, then treated with () or without () GDNF (30 ng/ml) for 30 min. Cleared cellular extracts (1 mg of protein/assay) were subjected to pull-down assays using immobilized GST fusion proteins containing the PTB domains of Dok-5 (amino acid residues 110–240), Dok-6 (amino acid residues 110–240), or GST as a control. Cellular protein complexes bound to the fusion proteins were eluted and subjected to SDS-PAGE followed by immunoblotting with Ret and phosphotyrosine (P-Tyr) antibodies. B, Dok-6 PTB domain binding to Ret requires the Ret Tyr1062 residue. CHP126 cells stably expressing wild type Ret9 or Ret51 isoforms (Ret9 and Ret51, respectively), isoforms containing tyrosine 1062 to phenylalanine (Y1062F) point mutations, or a kinase inactive Ret51 (Ret51 K758M) were treated and used in pull-down assays as described in A, above. Wild-type CHP126 cells (CHP) do not express endogenous Ret. The experiments in A and B were performed three times each with similar results. P-Ret, phospho-Tyr905 Ret; IB, immunoblotting.

Article Snippet: The sources and dilutions of primary antibodies used for immunoblot analyses were as follows: rabbit antiphosphotyrosine specific Ret Tyr905 (P-Ret) (43) was diluted 1:1000; rabbit polyclonal antibody against the extracellular domain of Ret (43) was diluted 1:1000; anti-HA, clone 12CA5 (Roche Applied Science) was diluted 1:1000 following reconstitution according to manufacturer’s protocol; mouse anti-phosphotyrosine (P-Tyr-100; Cell Signaling Technol- ogy, Beverly, MA).

Techniques: Residue, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Control, SDS Page, Western Blot, Binding Assay

Journal: Journal of Biological Chemistry

Article Title: Dok-6, a Novel p62 Dok Family Member, Promotes Ret-mediated Neurite Outgrowth

doi: 10.1074/jbc.m403726200

Figure Lengend Snippet: FIG. 6. Ret phosphorylates the C terminus of Dok-6 via Src. A, Dok-6 is a substrate of the Ret signaling cascade. N2A-1 cells were transiently transfected with expression vectors encoding HA-tagged versions of Dok-5 or Dok-6 or with an empty expression vector (Control). Two days after transfection, cells were deprived of serum for 1 h, then treated with () or without () GDNF (30 ng/ml) for 30 min. Cellular extracts were immunoprecipitated using anti-HA antibodies. The eluted immunocomplexes were then subjected to SDS-PAGE followed by HA and phosphotyrosine (P-Tyr) immunoblotting. B, the Dok-6 C terminus is tyrosine-phosphorylated in response to activation of the Ret signaling cascade. N2A-1 cells were transiently transfected with expression vectors encoding HA-tagged versions of Dok-6 or C-terminally truncated Dok-6 (amino acids 1–260, Dok-6C) or with an empty expression vector (Control). Two days after transfection, cells were treated as described above and anti-HA immunoprecipitates were subjected to SDS-PAGE followed by HA and P-Tyr immunoblotting. C, Ret phosphorylates Dok-6 through Src. N2A-1 cells were transiently transfected with an expression vector encoding HA-tagged Dok-6. Two days after transfection, cells were deprived of serum for 1 h, then treated with () or without () GDNF (30 ng/ml) for 30 min. Where indicated, cells were preincubated with either PP2 (1 M), PP3 (1 M), or SU6656 (SU, 2 M) for 30 min prior to GDNF stimulation. Anti-HA immunoprecipitates prepared from cellular extracts were subjected to SDS-PAGE followed by HA and P-Tyr immunoblotting. P-Ret, phospho-Tyr905 Ret; IP, immunoprecipitation; IB, immunoblotting.

Article Snippet: The sources and dilutions of primary antibodies used for immunoblot analyses were as follows: rabbit antiphosphotyrosine specific Ret Tyr905 (P-Ret) (43) was diluted 1:1000; rabbit polyclonal antibody against the extracellular domain of Ret (43) was diluted 1:1000; anti-HA, clone 12CA5 (Roche Applied Science) was diluted 1:1000 following reconstitution according to manufacturer’s protocol; mouse anti-phosphotyrosine (P-Tyr-100; Cell Signaling Technol- ogy, Beverly, MA).

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Immunoprecipitation, SDS Page, Western Blot, Activation Assay

Journal: Journal of Biological Chemistry

Article Title: Dok-6, a Novel p62 Dok Family Member, Promotes Ret-mediated Neurite Outgrowth

doi: 10.1074/jbc.m403726200

Figure Lengend Snippet: FIG. 7. Dok-6 promotes Ret-mediated neurite outgrowth. N2A-1 cells were infected with lentiviruses to permit the stable expression of HA-tagged Dok-5, Dok-6, C-terminally truncated Dok-6 (Dok-6C), or an empty expression cassette (Control). Virus infected cells selected by FACS against the Venus reporter gene were used in neurite outgrowth assays. A, expression of Dok proteins in lentivirus-infected N2A-1 cells. Cell extracts were immunoprecipitated using anti-HA antibodies. Immunoprecipitates were subjected to SDS-PAGE followed by HA immunoblotting (bottom panel). Ret activation is not affected by the expression of the Dok proteins used in this study (top panel). B, N2A-1 cells expressing the indicated Dok gene construct or empty cassette were placed in low serum medium (0.5% FBS), treated with () or without () GDNF (30 ng/ml) for 24 h, and photographed. C and D, quantification of neurite outgrowth. In C, cells were placed in low serum medium (0.5% FBS) and treated with the indicated concentration of GDNF for 24 h. Neurite outgrowth was assessed by evaluating cells in randomly chosen fields and calculating the percentage of neurite-bearing cells. Expression of Dok-6 significantly enhanced neurite outgrowth compared with N2A-1 cells expressing Dok-6C or an empty expression cassette (p 0.0001). There was no significant difference in neurite outgrowth induced by 5 ng/ml versus 30 ng/ml GDNF treatments within any given cell line, between Dok-6- and Dok-5-expressing N2A-1 cells, or between empty expression cassette and Dok-6C-expressing N2A-1 cells. In D, neurite lengths were measured in each cell line treated with 30 ng/ml GDNF for 24 h. Average neurite length in Dok-6-expressing N2A-1 cells was significantly greater than that of N2A-1 cells expressing Dok-6C or an empty expression cassette (p 0.002). Results in C and D were obtained from 3–5 independent experiments each. P-Ret, phospho-Tyr905 Ret; IP, immunoprecipitation; IB, immunoblotting.

Article Snippet: The sources and dilutions of primary antibodies used for immunoblot analyses were as follows: rabbit antiphosphotyrosine specific Ret Tyr905 (P-Ret) (43) was diluted 1:1000; rabbit polyclonal antibody against the extracellular domain of Ret (43) was diluted 1:1000; anti-HA, clone 12CA5 (Roche Applied Science) was diluted 1:1000 following reconstitution according to manufacturer’s protocol; mouse anti-phosphotyrosine (P-Tyr-100; Cell Signaling Technol- ogy, Beverly, MA).

Techniques: Infection, Expressing, Control, Virus, Immunoprecipitation, SDS Page, Western Blot, Activation Assay, Construct, Concentration Assay