Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Vasopressin increases S261 phosphorylation in AQP2-P262L, a mutant in recessive nephrogenic diabetes insipidus.

doi: 10.1093/ndt/gfs292

Figure Lengend Snippet: Fig. 1. The 30 kDa band of AQP2-P262L is due to phosphorylation. (A) Lysates from forskolin-treated MDCK cells transiently expressing AQP2- A190T or stably expressing wt-AQP2 and AQP2-P262L were treated with endo H or left untreated and subjected to immunoblotting for AQP2. Endo H treatment removed the high-mannose glycosylated (hm-glyc) form of AQP2-A190T, but did not affect the 30 kDa band of AQP2- P262L. (B) Lysate of MDCK cells stably expressing AQP2-P262L or wt- AQP2 were treated with phosphatase inhibitor (Phin), left untreated (C) or incubated with alkaline phosphatase (Ph). The 30 kDa band is removed after phosphatase treatment. Molecular masses of AQP2 proteins (A) or marker proteins (B) are indicated on the left (in kDa).

Article Snippet: Antibodies recognizing AQP2-pS261 were purchased from Novus Biological, Cambridge, UK.

Techniques: Phospho-proteomics, Expressing, Stable Transfection, Western Blot, Incubation, Marker

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Vasopressin increases S261 phosphorylation in AQP2-P262L, a mutant in recessive nephrogenic diabetes insipidus.

doi: 10.1093/ndt/gfs292

Figure Lengend Snippet: Fig. 2. Characterization of phospho-specific antibodies for AQP2. (A) Phospho-specific antibodies generated against peptides with phosphorylated S256, S264 and S269 or obtained from a company (S261) were checked for their reactivity against the different non-phosphorylated (np) and phosphorylated (p) peptides with dot blot analysis. All antibodies showed a reaction only against the phosphorylated peptide to which they were raised. (B) MDCK cells stably expressing hAQP2 or hAQP2-T269S were treated with indomethacine in the absence (I) or presence (IdDAVP) of dDAVP. Lysates were subjected to immunoblotting with the antibody raised against the pS269 peptide. The detected 29 kDa AQP2 band for both cell lines is intensified with forskolin, which revealed that our pS269 antibodies bind to pS269 as well as pT269. Marker proteins are indicated on the left (in kDa).

Article Snippet: Antibodies recognizing AQP2-pS261 were purchased from Novus Biological, Cambridge, UK.

Techniques: Generated, Dot Blot, Stable Transfection, Expressing, Western Blot, Marker

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Vasopressin increases S261 phosphorylation in AQP2-P262L, a mutant in recessive nephrogenic diabetes insipidus.

doi: 10.1093/ndt/gfs292

Figure Lengend Snippet: Fig. 3. Site-specific phosphorylation of AQP2 and AQP2-P262L. MDCK cells stably expressing AQP2 or AQP2-P262L were treated with indomethacine in the absence (I) or presence of forskolin (IF) and subjected to immunoblotting for total AQP2, or AQP2 phosphorylated at S256 (pS256), S261 (pS261) or T269 (pT269). Marker proteins are indicated on the left (in kDa).

Article Snippet: Antibodies recognizing AQP2-pS261 were purchased from Novus Biological, Cambridge, UK.

Techniques: Phospho-proteomics, Stable Transfection, Expressing, Western Blot, Marker

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Vasopressin increases S261 phosphorylation in AQP2-P262L, a mutant in recessive nephrogenic diabetes insipidus.

doi: 10.1093/ndt/gfs292

Figure Lengend Snippet: Fig. 4. Selective affinity of anti-pS261 antibodies for pS261 in wt- AQP2 and AQP2-P262L. Biotin-coupled peptides of the AQP2-C-tail (aa242–271) of wt-AQP2 and AQP2-P262l with (pS) or without phosphorylation at different sites were made and spotted on membranes. The membrane was incubated with anti-pS261 to show the affinity of the antibody or with streptavidin-HRP to show loading of equal peptide amounts.

Article Snippet: Antibodies recognizing AQP2-pS261 were purchased from Novus Biological, Cambridge, UK.

Techniques: Phospho-proteomics, Membrane, Incubation

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Vasopressin increases S261 phosphorylation in AQP2-P262L, a mutant in recessive nephrogenic diabetes insipidus.

doi: 10.1093/ndt/gfs292

Figure Lengend Snippet: Fig. 5. Expression and sorting of wt-AQP2, AQP2-S261A and AQP2-P262L in mpkCCD cells. mpkCCD cells stably expressing AQP2, AQP2- S261A or AQP2-P262L were treated with indomethacine in the absence (I) or presence of forskolin (IF). (A) Cells were fixed, stained with anti- AQP2 antibodies and subjected to confocal laser scanning microscopy to determine the AQP2 localization. (B) AQP2 immunoblot of lysates of these cells. Data from representative clones are shown. Marker proteins are indicated on the left (in kDa).

Article Snippet: Antibodies recognizing AQP2-pS261 were purchased from Novus Biological, Cambridge, UK.

Techniques: Expressing, Stable Transfection, Staining, Confocal Laser Scanning Microscopy, Western Blot, Clone Assay, Marker

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Vasopressin increases S261 phosphorylation in AQP2-P262L, a mutant in recessive nephrogenic diabetes insipidus.

doi: 10.1093/ndt/gfs292

Figure Lengend Snippet: Fig. 6. Effect of prevention of S261 phosphorylation on AQP2-P262L expression and sorting. mpkCCD cell stably expressing AQP2, AQP2- P262L or AQP2-S261A-P262L were treated with indomethacine in the absence (I) or presence of forskolin (IF). (A) Cell lysates were made and subjected to immunoblotting for AQP2. Marker proteins are indicated on the left (in kDa). (B) mpkCCD cells expressing AQP2-S261A-P262L were fixed, stained with anti-AQP2 antibodies and subjected to confocal laser scanning microscopy to determine AQP2 localization.

Article Snippet: Antibodies recognizing AQP2-pS261 were purchased from Novus Biological, Cambridge, UK.

Techniques: Phospho-proteomics, Expressing, Stable Transfection, Western Blot, Marker, Staining, Confocal Laser Scanning Microscopy

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Vasopressin increases S261 phosphorylation in AQP2-P262L, a mutant in recessive nephrogenic diabetes insipidus.

doi: 10.1093/ndt/gfs292

Figure Lengend Snippet: Fig. 7. Effect of S261 phosphorylation on the expression and sorting of wt-AQP2 and AQP2-P262L with fixed constitutive phosphorylation at S256/ S264/T269. mpkCCD cells stably expressing AQP2 or AQP2-P262L with fixed constitutive phosphorylation of S256, S264 and T269 (change into D/E) and mimicking S261 de-phosphorylation (S261A) or phosphorylation (S261D) were treated with indomethacine only (I) or together with forskolin (IF). (A) Cell lysates were subjected to immunoblotting for total AQP2. Marker proteins are indicated on the left (in kDa). (B) Cells with wt-AQP2 (left) or AQP2-P262L (right) constitutively de-phosphorylated at S261 (S261A) and treated with forskolin were subjected to AQP2 immunocytochemistry and confocal laser scanning microscopy to determine AQP2 localization.

Article Snippet: Antibodies recognizing AQP2-pS261 were purchased from Novus Biological, Cambridge, UK.

Techniques: Phospho-proteomics, Expressing, Stable Transfection, De-Phosphorylation Assay, Western Blot, Marker, Immunocytochemistry, Confocal Laser Scanning Microscopy