Journal: American Journal of Physiology - Renal Physiology

Article Title: Phosphatase inhibition increases AQP2 accumulation in the rat IMCD apical plasma membrane

doi: 10.1152/ajprenal.00150.2016

Figure Lengend Snippet: Effect of calyculin on total aquaporin 2 (AQP2) and pSAQP2 abundance in rat kidney inner medulla. Rat inner medullas were harvested after 30-min incubation with or without calyculin (5 μM) and analyzed by Western blot for total AQP2 and pS-AQP2 (arrows) as follows: (A) total AQP2, (B) pS256-AQP2, (C) pS261-AQP2, (D) pS264-AQP2, and (E) pS269-AQP2. Left: representative Western blots. The combined densitometry of total AQP2 and pSAQP2 (n = 8, means ± SE) is shown in the right graphs, respectively. Open bars, control; filled bars, calyculin treated. *P < 0.05, **P < 0.01.

Article Snippet: PVDF membranes were blocked for 60 min with 5% nonfat dry milk before overnight incubation with primary antibodies: our total AQP2 ( 12 , 24 ), pS 256 -AQP2 (Biorbyt, Burlington, NC; catalog no. orb317557), pS 261 -AQP2 (Avivasysbio, San Diego, CA; catalog no. OAPC00158), pS 264 -AQP2 (Thermo Fisher Scientific, Norcross, GA; catalog no. PA5-35387), pS 269 -AQP2 (Thermo Fisher Scientific; catalog no. PA5-35388), PP2A (R & D Systems, Minneapolis, MN; catalog no. AF1653), and PP2B (R & D Systems; catalog no. AF1348).

Techniques: Incubation, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Phosphatase inhibition increases AQP2 accumulation in the rat IMCD apical plasma membrane

doi: 10.1152/ajprenal.00150.2016

Figure Lengend Snippet: AQP2 expression in the cell membrane in rat kidney inner medulla. Rat inner medullas were incubated in calyculin (5 μM) for 30 min immediately after being harvested, and an inner medullary collecting duct (IMCD) suspension was prepared. The suspended IMCDs were then biotinylated, and the biotinylated protein pool was analyzed by Western blot for biotin-AQP2 (arrows) as follows: (A) AQP2, (B) pS256-AQP2, (C) pS264-AQP2, and (D) pre-bead lysis sample. Left: representative Western blots. Combined densitometry of the biotinylated samples is shown in the right graphs (n = 8, means ± SE). Open bars, control; filled bars, calyculin treated. **P < 0.01.

Article Snippet: PVDF membranes were blocked for 60 min with 5% nonfat dry milk before overnight incubation with primary antibodies: our total AQP2 ( 12 , 24 ), pS 256 -AQP2 (Biorbyt, Burlington, NC; catalog no. orb317557), pS 261 -AQP2 (Avivasysbio, San Diego, CA; catalog no. OAPC00158), pS 264 -AQP2 (Thermo Fisher Scientific, Norcross, GA; catalog no. PA5-35387), pS 269 -AQP2 (Thermo Fisher Scientific; catalog no. PA5-35388), PP2A (R & D Systems, Minneapolis, MN; catalog no. AF1653), and PP2B (R & D Systems; catalog no. AF1348).

Techniques: Expressing, Incubation, Western Blot, Lysis

Journal: American Journal of Physiology - Renal Physiology

Article Title: Phosphatase inhibition increases AQP2 accumulation in the rat IMCD apical plasma membrane

doi: 10.1152/ajprenal.00150.2016

Figure Lengend Snippet: Effect of tacrolimus on total AQP2 and pS-AQP2 abundance in rat kidney inner medulla. Rat inner medullas were harvested after 30-min incubation with or without tacrolimus (5 μM) and analyzed by Western blot for total AQP2 and pS-AQP2 (arrows) as follows: (A) total AQP2, (B) pS256-AQP2, (C) pS261-AQP2, (D) pS264-AQP2, and (E) pS269-AQP2. Left: representative Western blots. The combined densitometry of total AQP2 and pS-AQP2 (n = 8, means ± SE) is shown in the right graphs, respectively. Open bars, control; filled bars, calyculin treated. *P < 0.05.

Article Snippet: PVDF membranes were blocked for 60 min with 5% nonfat dry milk before overnight incubation with primary antibodies: our total AQP2 ( 12 , 24 ), pS 256 -AQP2 (Biorbyt, Burlington, NC; catalog no. orb317557), pS 261 -AQP2 (Avivasysbio, San Diego, CA; catalog no. OAPC00158), pS 264 -AQP2 (Thermo Fisher Scientific, Norcross, GA; catalog no. PA5-35387), pS 269 -AQP2 (Thermo Fisher Scientific; catalog no. PA5-35388), PP2A (R & D Systems, Minneapolis, MN; catalog no. AF1653), and PP2B (R & D Systems; catalog no. AF1348).

Techniques: Incubation, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Phosphatase inhibition increases AQP2 accumulation in the rat IMCD apical plasma membrane

doi: 10.1152/ajprenal.00150.2016

Figure Lengend Snippet: AQP2 expression in the cell membrane in rat kidney inner medulla. Rat inner medullas were incubated in tacrolimus (5 μM) for 30 min immediately after being harvested, and an IMCD suspension was prepared. The suspended IMCDs were then biotinylated, and the biotinylated protein pool was analyzed by Western blot for biotin-AQP2 (arrows) as follows: (A) AQP2 and (B) pre-bead lysis sample. Left: representative Western blots. Combined densitometry of the biotinylated samples is shown in the right graphs (n = 8, means ± SE). Open bars, control; filled bars, calyculin treated.

Article Snippet: PVDF membranes were blocked for 60 min with 5% nonfat dry milk before overnight incubation with primary antibodies: our total AQP2 ( 12 , 24 ), pS 256 -AQP2 (Biorbyt, Burlington, NC; catalog no. orb317557), pS 261 -AQP2 (Avivasysbio, San Diego, CA; catalog no. OAPC00158), pS 264 -AQP2 (Thermo Fisher Scientific, Norcross, GA; catalog no. PA5-35387), pS 269 -AQP2 (Thermo Fisher Scientific; catalog no. PA5-35388), PP2A (R & D Systems, Minneapolis, MN; catalog no. AF1653), and PP2B (R & D Systems; catalog no. AF1348).

Techniques: Expressing, Incubation, Western Blot, Lysis

Journal: American Journal of Physiology - Renal Physiology

Article Title: Phosphatase inhibition increases AQP2 accumulation in the rat IMCD apical plasma membrane

doi: 10.1152/ajprenal.00150.2016

Figure Lengend Snippet: Immunostaining for AQP2 and pSAQP2 in rat kidney inner medullas. Representative images (original magnification ×400) of immunostaining of rat inner medullary samples of control group (top), calyculin-treated group (middle), and tacrolimus-treated group (bottom) with AQP2, pS256-AQP2, pS261-AQP2, and pS264-AQP2. The apical membrane abundance of AQP2, pS256-AQP2, and pS264-AQP2 is increased in the calyculin- and tacrolimus-treated groups compared with the control group samples.

Article Snippet: PVDF membranes were blocked for 60 min with 5% nonfat dry milk before overnight incubation with primary antibodies: our total AQP2 ( 12 , 24 ), pS 256 -AQP2 (Biorbyt, Burlington, NC; catalog no. orb317557), pS 261 -AQP2 (Avivasysbio, San Diego, CA; catalog no. OAPC00158), pS 264 -AQP2 (Thermo Fisher Scientific, Norcross, GA; catalog no. PA5-35387), pS 269 -AQP2 (Thermo Fisher Scientific; catalog no. PA5-35388), PP2A (R & D Systems, Minneapolis, MN; catalog no. AF1653), and PP2B (R & D Systems; catalog no. AF1348).

Techniques: Immunostaining

Journal: American Journal of Physiology - Renal Physiology

Article Title: Role of PKC and AMPK in hypertonicity-stimulated water reabsorption in rat inner medullary collecting ducts

doi: 10.1152/ajprenal.00491.2017

Figure Lengend Snippet: Hypertonicity increased phosphorylation of aquaporin-2 (AQP2) at serine 256 in rat inner medulla (IM). A: representative Western blot showing AQP2 abundance and phosphorylated AQP2 in rat IM with hypertonic (Hyper) stimulation (690 mosmol/kgH2O) for 15 min. Brackets indicate AQP2 glycosylated protein between 35 and 45 kDa and arrows indicate unglycosylated protein at 29 kDa. Samples from a different rat were loaded into each lane. B: bar graph showing the ratio of the band densities of phosphorylated AQP2 to total AQP2 abundance. Bars = means ± SE. *P < 0.05 vs. isotonic (Iso) treatment (290 mosmol/kgH2O); n = 4 rats/condition.

Article Snippet: Antibodies to AQP2, AQP2 phosphorylated at serine 256 or 261, AMPK, and pThr172-AMPK were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Phospho-proteomics, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Role of PKC and AMPK in hypertonicity-stimulated water reabsorption in rat inner medullary collecting ducts

doi: 10.1152/ajprenal.00491.2017

Figure Lengend Snippet: Hypertonicity decreased phosphorylation of aquaporin-2 (AQP2) at serine 261 in rat inner medulla (IM). A: representative Western blot showing AQP2 abundance and phosphorylated AQP2 in rat IM with hypertonic (Hyper) stimulation (690 mosmol/kgH2O) for 15 min. Brackets indicate AQP2 glycosylated protein between 35 and 45 kDa and arrows indicate unglycosylated protein at 29 kDa. Samples from a different rat were loaded into each lane. B: bar graph showing the ratio of the band densities of phosphorylated AQP2 to total AQP2 abundance. Bars = means ± SE. *P < 0.05 vs. isotonic (Iso) treatment (290 mosmol/kgH2O); n = 7 rats/condition.

Article Snippet: Antibodies to AQP2, AQP2 phosphorylated at serine 256 or 261, AMPK, and pThr172-AMPK were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Phospho-proteomics, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Role of PKC and AMPK in hypertonicity-stimulated water reabsorption in rat inner medullary collecting ducts

doi: 10.1152/ajprenal.00491.2017

Figure Lengend Snippet: Inhibition of protein kinase C (PKC) with chelerythrine (Chel) did not change phosphorylation of aquaporin-2 (AQP2) at serine 256 in rat inner medulla (IM). A: representative Western blot showing AQP2 abundance and phosphorylated AQP2 in rat IM with hypertonic (Hyper) stimulation (690 mosmol/kgH2O) and treatment of 10 µM chelerythrine (PKC inhibitor) for 15 min. Brackets indicate AQP2 glycosylated protein between 35 and 45 kDa and arrows indicate unglycosylated protein at 29 kDa. Samples from a different rat were loaded into each lane. B: bar graph showing the ratio of the band densities of phosphorylated AQP2 to total AQP2 abundance. Bars = means ± SE; results were not significantly different between hypertonic treatment alone and combination of hypertonicity and chelerythrine; n = 4 rats/condition.

Article Snippet: Antibodies to AQP2, AQP2 phosphorylated at serine 256 or 261, AMPK, and pThr172-AMPK were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Inhibition, Phospho-proteomics, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Role of PKC and AMPK in hypertonicity-stimulated water reabsorption in rat inner medullary collecting ducts

doi: 10.1152/ajprenal.00491.2017

Figure Lengend Snippet: Inhibition of protein kinase C with chelerythrine (Chel) increased phosphorylation of aquaporin-2 (AQP2) at serine 261 in rat inner medulla (IM). A: representative Western blot showing AQP2 abundance and phosphorylated AQP2 in rat IM with hypertonic (Hyper) stimulation (690 mosmol/kgH2O) and treatment of 10 µM chelerythrine for 15 min. Brackets indicate AQP2 glycosylated protein between 35 and 45 kDa and arrows indicate unglycosylated protein at 29 kDa. Samples from a different rat were loaded into each lane. B: bar graph showing the ratio of the band densities of phosphorylated AQP2 to total AQP2 abundance. Bars = means ± SE. *P < 0.01 vs. hypertonic treatment (690 mosmol/kgH2O) only; n = 4 rats/condition.

Article Snippet: Antibodies to AQP2, AQP2 phosphorylated at serine 256 or 261, AMPK, and pThr172-AMPK were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Inhibition, Phospho-proteomics, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Role of PKC and AMPK in hypertonicity-stimulated water reabsorption in rat inner medullary collecting ducts

doi: 10.1152/ajprenal.00491.2017

Figure Lengend Snippet: Inhibition of adenosine monophosphate kinase (AMPK) with Compound C (Comp C) did not change phosphorylation of aquaporin-2 (AQP2) at serine 256 in rat inner medulla (IM). A: representative Western blot showing AQP2 abundance and phosphorylated AQP2 in rat IM with hypertonic (Hyper) stimulation (690 mosmol/kgH2O) and treatment of 10 µM Compound C (AMPK inhibitor) for 15 min. Brackets indicate AQP2 glycosylated protein between 35 and 45 kDa and arrows indicate unglycosylated protein at 29 kDa. Samples from a different rat were loaded into each lane. B: bar graph showing the ratio of the band densities of phosphorylated AQP2 to total AQP2 abundance. Bars = means ± SE; results were not significantly different between hypertonic treatment alone and combination of hypertonicity and compound C; n = 4 rats/condition.

Article Snippet: Antibodies to AQP2, AQP2 phosphorylated at serine 256 or 261, AMPK, and pThr172-AMPK were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Inhibition, Phospho-proteomics, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Role of PKC and AMPK in hypertonicity-stimulated water reabsorption in rat inner medullary collecting ducts

doi: 10.1152/ajprenal.00491.2017

Figure Lengend Snippet: Inhibition of adenosine monophosphate kinase (AMPK) with Compound C (Comp C) did not change phosphorylation of aquaporin-2 (AQP2) at serine 261 in rat inner medulla (IM). A: representative Western blot showing AQP2 abundance and phosphorylated AQP2 in rat IM with hypertonic (Hyper) stimulation (690 mosmol/kgH2O) and treatment of 10 µM Compound C (AMPK inhibitor) for 15 min. Brackets indicate AQP2 glycosylated protein between 35 and 45 kDa and arrows indicate unglycosylated protein at 29 kDa. Samples from a different rat were loaded into each lane. B: bar graph showing the ratio of the band densities of phosphorylated AQP2 to total AQP2 abundance. Bars = means ± SE; results were not significantly different between hypertonic treatment alone and combination of hypertonicity and Compound C; n = 4 rats/condition.

Article Snippet: Antibodies to AQP2, AQP2 phosphorylated at serine 256 or 261, AMPK, and pThr172-AMPK were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Inhibition, Phospho-proteomics, Western Blot

Journal: American Journal of Physiology - Renal Physiology

Article Title: Role of PKC and AMPK in hypertonicity-stimulated water reabsorption in rat inner medullary collecting ducts

doi: 10.1152/ajprenal.00491.2017

Figure Lengend Snippet: Stimulation of adenosine monophosphate kinase with AICAR increased phosphorylation of aquaporin-2 (AQP2) at serine 256 in rat inner medulla (IM). A: representative Western blot showing AQP2 abundance and phosphorylated AQP2 in rat IM with AICAR stimulation for 15 min. Brackets indicate AQP2 glycosylated protein between 35 and 45 kDa and arrows indicate unglycosylated protein at 29 kDa. Samples from a different rat were loaded into each lane. B: bar graph showing the ratio of the band densities of phosphorylated AQP2 to total AQP2 abundance. Bars = means ± SE. *P < 0.05 vs. control tissues (290 mosmol/kgH2O, without AICAR treatment); n = 4 rats/condition.

Article Snippet: Antibodies to AQP2, AQP2 phosphorylated at serine 256 or 261, AMPK, and pThr172-AMPK were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: American Journal of Physiology - Renal Physiology

Article Title: Role of PKC and AMPK in hypertonicity-stimulated water reabsorption in rat inner medullary collecting ducts

doi: 10.1152/ajprenal.00491.2017

Figure Lengend Snippet: Stimulation of adenosine monophosphate kinase with AICAR did not change phosphorylation of aquaporin-2 (AQP2) at serine 261 in rat inner medulla (IM). A: representative Western blot showing AQP2 abundance and phosphorylated AQP2 in rat IM with AICAR stimulation for 15 min. Brackets indicate AQP2 glycosylated protein between 35 and 45 kDa and arrows indicate unglycosylated protein at 29 kDa. Samples from a different rat were loaded into each lane. B: bar graph showing the ratio of the band densities of phosphorylated AQP2 to total AQP2 abundance. Bars = means ± SE; results were not significantly different between control and AICAR-treated tissues; n = 4 rats/condition.

Article Snippet: Antibodies to AQP2, AQP2 phosphorylated at serine 256 or 261, AMPK, and pThr172-AMPK were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: American Journal of Physiology - Renal Physiology

Article Title: Role of PKC and AMPK in hypertonicity-stimulated water reabsorption in rat inner medullary collecting ducts

doi: 10.1152/ajprenal.00491.2017

Figure Lengend Snippet: Protein kinase C (PKC) and adenosine monophosphate kinase (AMPK) did not change phosphorylation of AQP2 at serine 256 in rat inner medulla (IM). A: representative Western blot showing AQP2 abundance and phosphorylated AQP2 in rat IM with phorbol dibutyrate (PDBu) (PKC activator), metformin (Met; AMPK stimulator), or both for 30 min. Brackets indicate AQP2 glycosylated protein between 35 and 45 kDa and arrows indicate unglycosylated protein at 29 kDa. Samples from a different rat were loaded into each lane. B: bar graph showing the ratio of the band densities of phosphorylated AQP2 to total AQP2 abundance. Bars = means ± SE; results were not significantly different from the isotonic control group without PDBu and metformin. n = 6 rats/condition. Ctrl, control.

Article Snippet: Antibodies to AQP2, AQP2 phosphorylated at serine 256 or 261, AMPK, and pThr172-AMPK were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: American Journal of Physiology - Renal Physiology

Article Title: Role of PKC and AMPK in hypertonicity-stimulated water reabsorption in rat inner medullary collecting ducts

doi: 10.1152/ajprenal.00491.2017

Figure Lengend Snippet: Protein kinase C and adenosine monophosphate kinase decreased phosphorylation of aquaporin-2 (AQP2) at serine 261, but combined treatment did not provide an additive effect in rat inner medulla (IM). A: representative Western blot showing AQP2 abundance and phosphorylated AQP2 in rat IM with phorbol dibutyrate (PDBu), metformin (Met), or both for 30 min. Brackets indicate AQP2 glycosylated protein between 35 and 45 kDa and arrows indicate unglycosylated protein at 29 kDa. Samples from a different rat were loaded into each lane. B: bar graph showing the ratio of the band densities of phosphorylated AQP2 to total AQP2 abundance. Bars = means ± SE; *P < 0.01 vs. isotonic control groups without PDBu and metformin; n = 6 rats/condition. Ctrl, control.

Article Snippet: Antibodies to AQP2, AQP2 phosphorylated at serine 256 or 261, AMPK, and pThr172-AMPK were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Phospho-proteomics, Western Blot, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Systems-level identification of PKA-dependent signaling in epithelial cells

doi: 10.1073/pnas.1709123114

Figure Lengend Snippet: AQP2 abundance in PKA knockout cells and its rescue by PKA. All observations were made in the presence of dDAVP (0.1 nM continuously). (A, D, and G) Western blots for PKA-Cα, PKA-Cβ, and AQP2 are shown for 12 control clones versus 12 PKA-Cα knockout clones (A), 13 control clones versus 11 PKA-Cβ knockout clones (D), and 12 control clones versus 12 PKA dKO (G). Loading: PKA-Cα blots, 20 μg; PKA-Cβ blots, 30 μg; AQP2 blots, 10 μg; Coomassie, 7 μg. G, glycosylated; nG, nonglycosylated. (B, C, E, F, and H) Band density quantification of the respective immunoblots using beeswarm plus boxplot visualization. Each point is a quantification of a single lane. The heavy horizontal lines represent the median. Band density for AQP2 is summed for glycosylated and nonglycosylated bands. Band densities were normalized by the mean of respective control observations. (I) Immunofluorescence images showing that clusters of PKA dKO cells transfected with PKA-Cα or PKA-Cβ plasmids express AQP2 protein. DAPI labeling shows that the cells are confluent. (Scale bars, 30 μm.) (J and K) Western blot (J) and quantification (K) of protein abundance in transfected cells (n = 3, mean ± SD, *P < 0.05). Values are normalized by band density of AQP2 in vasopressin-treated control cells and expressed as a percentage. Loading: PKA-Cα blots, 20 μg; PKA-Cβ blots, 30 μg; AQP2 blots, 60 μg; Coomassie, 7 μg.

Article Snippet: Control and PKA dKO cells were transfected with a plasmid vector to express AQP2 (Sino Biological; MG57478-UT) and immediately seeded on permeable supports and grown to confluence in the absence of vasopressin (4 to 5 d). dDAVP (0.1 nM) was added to the basolateral medium before cells were prepared for immunofluorescence and Western blotting.

Techniques: Knock-Out, Western Blot, Clone Assay, Immunofluorescence, Transfection, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Systems-level identification of PKA-dependent signaling in epithelial cells

doi: 10.1073/pnas.1709123114

Figure Lengend Snippet: RNA-seq–based transcriptomic and SILAC-based quantitative proteomic analysis of PKA dKO versus control cells. (A) Distribution of RNA-seq reads across gene bodies of selected genes for one PKA dKO/control pair: Aqp2 (decreased), Prkaca (decreased), Prkacb (unchanged), Avpr2 (unchanged), Marcks (increased), and Rhcg (increased). Reading direction: blue, left to right; red, right to left. The vertical axis shows binned read counts normalized by total read number (read counts per 107). (B) Volcano plot for 10,190 transcripts expressed in control and PKA dKO cells, quantified for three pairs of PKA dKO vs. control clones. Red points are transcripts with FDR <0.05. Labeled transcripts are discussed in the text. (C) MS1 spectra of representative β-actin, PKA-Cα, PKA-Cβ, and AQP2 peptides showing peaks for control cells (labeled with heavy amino acids) and PKA dKO cells (labeled with light amino acids). Dashed brackets indicate the expected (but not observed) peak locations in the PKA dKO cells (light). The vertical axis shows peptide ion intensity, and the horizontal axis shows the mass-to-charge ratio (m/z). Each peptide has several m/z peaks due to the presence of natural isotopes. (D) The distribution of protein abundance changes for 7,647 proteins in PKA dKO versus control cells (n = 3 pairs). The horizontal axis shows the median log2 (dKO/Ctrl) over the three determinations. Vertical dashed lines indicate mean and ±2 SD (mean, −0.02; SD, 0.87). (E) Volcano plot for the 7,647 proteins quantified in all three pairs of samples. Red points indicate proteins with FDR <0.05. Proteins with FDR <0.05 and absolute value of log2 (dKO/Ctrl) >2 are labeled with the official gene symbol. (F) Correlation between changes in transcript abundance (RNA-seq) and protein abundance (SILAC) in PKA dKO versus control cells. Genes with FDR <0.05 in both analyses are labeled with the official gene symbol. The blue line shows linear regression (with ±SE in gray) calculated from all data.

Article Snippet: Control and PKA dKO cells were transfected with a plasmid vector to express AQP2 (Sino Biological; MG57478-UT) and immediately seeded on permeable supports and grown to confluence in the absence of vasopressin (4 to 5 d). dDAVP (0.1 nM) was added to the basolateral medium before cells were prepared for immunofluorescence and Western blotting.

Techniques: RNA Sequencing Assay, Clone Assay, Labeling

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Systems-level identification of PKA-dependent signaling in epithelial cells

doi: 10.1073/pnas.1709123114

Figure Lengend Snippet: Phosphorylation of aquaporin-2 in PKA dKO versus control mpkCCD cells. (A) AQP2 membrane-spanning topology. AQP2 has six transmembrane domains. A cluster of four vasopressin-dependent phosphorylation sites is present within the terminal 16 amino acids in the C-terminal tail. P, phosphorylation site. (B) The vasopressin-regulated phosphorylation sites are shown. Sequences surrounding Ser256, Ser264, and Ser269 are compatible with phosphorylation by basophilic protein kinases. Ser261 has a proline in position +1 and is presumably phosphorylated by a member of the MAPK family. Vasopressin decreases phosphorylation of Ser261 and increases phosphorylation at the other three sites. (C) Effect of PKA dKO on AQP2 phosphorylation levels. Both control and PKA dKO cells were transfected with AQP2, grown on a solid substratum for 24 h, and then treated with the adenylyl cyclase activator forskolin for 30 min (n = 3). Western blotting was done with phospho-specific antibodies recognizing each of the four phosphorylation sites (Upper) and quantified by densitometry (Lower). The bar graphs show normalized abundances as mean ± SD. Total AQP2 was quantified with a non–phospho-specific AQP2 antibody. (D) Low-power immunofluorescence images of total and phosphorylated AQP2 in PKA dKO and control cells. Cells were transfected with AQP2 and grown on a permeable support without dDAVP for 4 d. Subsequently, cells were stimulated with 0.1 nM dDAVP for 30 min. (Scale bars, 50 μm.)

Article Snippet: Control and PKA dKO cells were transfected with a plasmid vector to express AQP2 (Sino Biological; MG57478-UT) and immediately seeded on permeable supports and grown to confluence in the absence of vasopressin (4 to 5 d). dDAVP (0.1 nM) was added to the basolateral medium before cells were prepared for immunofluorescence and Western blotting.

Techniques: Transfection, Western Blot, Immunofluorescence

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Systems-level identification of PKA-dependent signaling in epithelial cells

doi: 10.1073/pnas.1709123114

Figure Lengend Snippet: Phosphorylation of AQP2 in PKA single-KO cells. PKA single-KO cells were grown on membrane supports in the presence of dDAVP (0.1 nM) to assure high levels of endogenous AQP2. dDAVP was withdrawn for a 2-h incubation and then was readded at 0.1 nM dDAVP for 30 min. Loading volume was adjusted to be the same total AQP2 to allow direct comparison of phosphorylation. n = 3, mean ± SD. (A) PKA-Cα single-knockout cells. (B) PKA-Cβ single-knockout cells. Bars represent normalized band densities (normalized by mean value for control cells in the absence of dDAVP).

Article Snippet: Control and PKA dKO cells were transfected with a plasmid vector to express AQP2 (Sino Biological; MG57478-UT) and immediately seeded on permeable supports and grown to confluence in the absence of vasopressin (4 to 5 d). dDAVP (0.1 nM) was added to the basolateral medium before cells were prepared for immunofluorescence and Western blotting.

Techniques: Incubation, Knock-Out

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Systems-level identification of PKA-dependent signaling in epithelial cells

doi: 10.1073/pnas.1709123114

Figure Lengend Snippet: PKA signaling mapped to functional effects of vasopressin. (A) Direct PKA targets and their physiological and functional effects. (B–H) PKA-regulated signaling network in MAP kinase signaling (B), decreased apoptosis (C), Aqp2 gene transcription (D), actin dynamics (E), AQP2 phosphorylation (F), exocytosis (G), AQP2 endocytosis (H), and AQP2 protein stability (H). Data sources are given at https://hpcwebapps.cit.nih.gov/ESBL/PKANetwork/.

Article Snippet: Control and PKA dKO cells were transfected with a plasmid vector to express AQP2 (Sino Biological; MG57478-UT) and immediately seeded on permeable supports and grown to confluence in the absence of vasopressin (4 to 5 d). dDAVP (0.1 nM) was added to the basolateral medium before cells were prepared for immunofluorescence and Western blotting.

Techniques: Functional Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Systems-level identification of PKA-dependent signaling in epithelial cells

doi: 10.1073/pnas.1709123114

Figure Lengend Snippet: Role of PKA in nuclear translocation of transcriptional regulators, histone acetylation, actin polymerization, and apical membrane trafficking of AQP2. (A) Nuclear translocation of transcriptional regulators in response to vasopressin. Western blot of nuclear and cytoplasmic extracts of various transcriptional regulators (Left). Densitometric analysis showing mean and SD (Right). CE, cytoplasmic extract; NE, nuclear extract. (B) Distribution of ChIP-seq reads across gene bodies of selected genes for vehicle- or dDAVP-treated cells. Green boxes highlight two genomic regions in which increases were observable. (C) Confocal projection x–y (Top) and x–z (Bottom thin panels) images showing changes in actin polymerization in response to vasopressin. Alexa-594 phalloidin staining in cells treated with vehicle or dDAVP. (Scale bars, 10 μm.) (D) Vasopressin-dependent AQP2 trafficking to the apical plasma membrane in control and PKA dKO cells. Confocal x–y (Top) and x–z (Bottom thin panels) images of cells treated with vehicle or dDAVP using anti-AQP2 antibody (green). DAPI-stained nuclei, blue. (Scale bars, 10 μm.)

Article Snippet: Control and PKA dKO cells were transfected with a plasmid vector to express AQP2 (Sino Biological; MG57478-UT) and immediately seeded on permeable supports and grown to confluence in the absence of vasopressin (4 to 5 d). dDAVP (0.1 nM) was added to the basolateral medium before cells were prepared for immunofluorescence and Western blotting.

Techniques: Translocation Assay, Western Blot, ChIP-sequencing, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Systems-level identification of PKA-dependent signaling in epithelial cells

doi: 10.1073/pnas.1709123114

Figure Lengend Snippet: Materials

Article Snippet: Control and PKA dKO cells were transfected with a plasmid vector to express AQP2 (Sino Biological; MG57478-UT) and immediately seeded on permeable supports and grown to confluence in the absence of vasopressin (4 to 5 d). dDAVP (0.1 nM) was added to the basolateral medium before cells were prepared for immunofluorescence and Western blotting.

Techniques: Recombinant, Transfection, Electron Microscopy, Mass Spectrometry, Bicinchoninic Acid Protein Assay, Purification, Chromatin Immunoprecipitation, Magnetic Beads, Expressing, Plasmid Preparation, Sequencing, CRISPR, Software, Microscopy

Journal: Physiological Reports

Article Title: TRPV4 functional status in cystic cells regulates cystogenesis in autosomal recessive polycystic kidney disease during variations in dietary potassium

doi: 10.14814/phy2.15641

Figure Lengend Snippet: TRPV4 activity regulates subcellular AQP2 distribution in cystic cells of PCK453 rats. (a) Representative confocal images showing AQP2 (pseudocolor red) distribution in kidney sections of PCK453 rats fed regular (0.9%K + ), high KCl (5%K + ), and high KB/C (5%K + , bicarbonate: citrate as 4:1) diets for 1 month. Nuclear Dapi staining is shown with pseudocolor blue. Areas with cystic (1) and non‐dilated collecting duct (2) are shown below at higher magnification. The averaged intensities of AQP2‐reporting fluorescent signals around the apical area in cystic (b) and non‐dilated collecting duct (c) cells from the conditions in (a). For each individual cell the fluorescent signals were normalized to their corresponding maximal value.

Article Snippet: Sections were incubated overnight at +4°C with anti‐AQP2‐ATTO Fluor‐550 (1:200, Alomone Labs; Cat. # AQP2‐002‐AO) and anti‐TRPV4 (1:500, Alomone labs; Cat.#.

Techniques: Activity Assay, Staining

Journal: Physiological Reports

Article Title: TRPV4 functional status in cystic cells regulates cystogenesis in autosomal recessive polycystic kidney disease during variations in dietary potassium

doi: 10.14814/phy2.15641

Figure Lengend Snippet: TRPV4 activity is inversely related to cAMP levels in cystic cells of PCK453 rats. Summary graph comparing dispersion (decrease by 50% from maximum) of AQP2‐reporting signal in non‐dilated collecting duct versus cystic cells in kidney sections of PCK453 rats fed regular (0.9%K + ), high KCl (5%K + ), and high KB/C (5%K + , bicarbonate: citrate as 4:1) diets for 1 month. Bars and whiskers represent SE and SD, respectively. Mean and median values are denoted with lines. Numbers of each experimental groups are shown below. Kidney sections from at least 4 different animals were used for each tested group. *Significant changes ( p < 0.05, one‐way ANOVA with post hoc Tukey test) between groups shown with brackets on the top.

Article Snippet: Sections were incubated overnight at +4°C with anti‐AQP2‐ATTO Fluor‐550 (1:200, Alomone Labs; Cat. # AQP2‐002‐AO) and anti‐TRPV4 (1:500, Alomone labs; Cat.#.

Techniques: Activity Assay, Dispersion

Journal: Nature Communications

Article Title: Endocycle-related tubular cell hypertrophy and progenitor proliferation recover renal function after acute kidney injury

doi: 10.1038/s41467-018-03753-4

Figure Lengend Snippet: Only a small TEC subset proliferates after AKI and partially replaces lost TECs. a GFR in ischemic mice ( n = 13) normalized on the GFR at baseline and on sham-operated control group ( n = 5). One-way ANOVA post hoc Tukey. b , c Juxtaposed images of a Pax8/Confetti mouse kidney at day 0 (T0, n = 5) ( b ) and 30 after IRI (IRI T30, n = 4) ( c ). Arrows indicate single-colored clones. OSOM outer stripe of outer medulla; ISOM inner stripe of outer medulla. d Number of Pax8+, Pax8+AQP2−, and Pax8+AQP1+ cells in Pax8/Confetti mice at day 0 (T0, white column, n = 5) and at day 30 after IRI (IRI T30, gray column, n = 4). Mann–Whitney test. * p < 0.05, ** p < 0.01 IRI T30 vs. T0. e BUN in healthy Pax8/Confetti mice ( n = 5) and in Pax8/Confetti mice after nephrotoxic AKI ( n = 6). One-way ANOVA post hoc Tukey * p < 0.05 glycerol-treated mice vs. healthy. f GFR in Pax8/Confetti mice after nephrotoxic AKI ( n = 7) normalized on the GFR at baseline and on healthy mice ( n = 5). One-way ANOVA post hoc Tukey. g Number of Pax8+ cells in Pax8/Confetti mice at day 0 (T0, white column, n = 5) and day 30 after nephrotoxic AKI (Gly T30, gray with sparse pattern column, n = 4). Mann–Whitney test * p < 0.05 Gly T30 vs. T0. h Number of Pax8+AQP2− cells in Pax8/Confetti mice at day 0 (T0, white column, n = 5) and day 30 after nephrotoxic AKI (Gly T30, gray with sparse pattern column, n = 4). Mann–Whitney test * p < 0.05 Gly T30 vs. T0. i Single-colored clones in AQP2− tubules in Pax8/Confetti mice at day 30 after IRI ( n = 4). AQP2 staining is white. j Clone frequency analysis of Pax8+AQP2− cells in Pax8/Confetti mice at day 0 (T0, n = 5), in age-matched controls (T30, n = 4), in sham-operated mice ( n = 4), at day 30 after IRI (IRI T30, n = 4) and at day 30 after nephrotoxic AKI (Gly T30, n = 4). Mann–Whitney test * p < 0.05 IRI T30 vs. T0, T30, sham and Gly T30 vs. T0 and T30. k Percentage of Pax8+AQP2− cells in Pax8/Confetti mice at day 0 (T0, n = 5), at day 30 after IRI (IRI T30, n = 4) and at day 30 after nephrotoxic AKI (Gly T30, n = 4). Mann–Whitney test * p < 0.05 IRI T30 and Gly T30 vs. T0. l Percentage of Pax8+AQP2− clonogenic cells (gray column) in Pax8/Confetti mice at day 30 after IRI (IRI T30, n = 4) and at day 30 after nephrotoxic AKI (Gly T30, n = 4) vs. day 0 ( n = n = 5). The white column is the percentage of Pax8+AQP2− that did not generate clones. Data are mean ± SEM. Scale bars 40 µm. Pax8+ = Pax8 lineage-positive cells

Article Snippet: The remaining part of the cells were incubated with anti-AQP2 pAb (bs-4611R, dilution 1:20, Bioss Inc.) followed by anti-rabbit MicroBeads (Miltenyi Biotec S.r.l.), then passed through MS columns (Miltenyi Biotec S.r.l.) accordingly with the manufacturer’s protocol and collecting the first flow-through containing unlabeled cells in order to perform a magnetic cell depletion of AQP2+ cells.

Techniques: Clone Assay, MANN-WHITNEY, Staining

Journal: Nature Communications

Article Title: Endocycle-related tubular cell hypertrophy and progenitor proliferation recover renal function after acute kidney injury

doi: 10.1038/s41467-018-03753-4

Figure Lengend Snippet: Kidney tubules contain a distinct, predefined Pax2 lineage-positive tubular cell subset. a – c Juxtaposed confocal images of a kidney section from cortex to inner stripe of outer medulla in adult Pax2/Confetti mice ( n = 4). Confetti reporter shows Pax2 lineage-positive single cells scattered within the proximal tubules, distal tubules and collecting ducts as demonstrated by immunolabelling for AQP1 ( a ), THP ( b ) and AQP2 ( c ), respectively (white). OSOM outer stripe of outer medulla; ISOM inner stripe of outer medulla. Scale bars 40 µm. d Schematic localization of Pax2− lineage-positive cells (red dots) in the proximal tubule, stained by AQP1 (light gray), in the thick ascending limb and in the distal tubule, stained by THP (dark gray), of the nephron. e – h Representative images of a kidney section in healthy Pax2/Confetti mice showing ( n = 4) the distribution of Pax2 lineage-positive cells in S3 segment of proximal tubules (AQP1+, white) ( e ), in thick ascending limbs and distal tubules (THP+, white) ( f ), in collecting ducts (AQP2+, white) ( g ) and in S1–S2 segment of proximal tubules (Megalin+, white) ( h ). Scale bars 20 µm. i Experimental schemes. j , k Representative images of a kidney section in the OSOM of Pax2/Confetti mice showing Pax2+AQP2− cells after 30 days of washout (T30, n = 5) ( j ) and after 30 days with doxycycline (doxycycline T30, n = 4) ( k ). AQP2 staining is white. l Number of Pax2+AQP2− cells in OSOM of Pax2/Confetti mice at T30 ( n = 5) and after 30 days with doxycycline (doxy T30, n = 4). Mann–Whitney test NS. Data are mean ± SEM. Scale bars 20 µm. Pax2+ = Pax2 lineage-positive cells

Article Snippet: The remaining part of the cells were incubated with anti-AQP2 pAb (bs-4611R, dilution 1:20, Bioss Inc.) followed by anti-rabbit MicroBeads (Miltenyi Biotec S.r.l.), then passed through MS columns (Miltenyi Biotec S.r.l.) accordingly with the manufacturer’s protocol and collecting the first flow-through containing unlabeled cells in order to perform a magnetic cell depletion of AQP2+ cells.

Techniques: Staining, MANN-WHITNEY

Journal: Nature Communications

Article Title: Endocycle-related tubular cell hypertrophy and progenitor proliferation recover renal function after acute kidney injury

doi: 10.1038/s41467-018-03753-4

Figure Lengend Snippet: Pax2 lineage-positive cells show increased survival and proliferative capacity in comparison to other TECs after AKI. a GFR in Pax2/Confetti mice after ischemic AKI ( n = 9) normalized on the GFR at baseline and on sham-operated control group ( n = 5). One-way ANOVA post hoc Tukey. b Juxtaposed confocal images of a kidney section in Pax2/Confetti mice at day 30 after IRI ( n = 5). Arrows indicate single colored clones. Scale bars 40 µm. c , d Single colored clones in AQP2− tubules in Pax2/Confetti mice at day 30 after IRI ( n = 5). AQP2 staining is white. Scale bars 20 µm. e Number of Pax2+AQP2− TECs in Pax2/Confetti mice at day 0 (T0, n = 4), in age-matched controls (T30; n = 5), in sham-operated mice (sham; n = 5) and at day 30 after IRI (IRI T30, n = 5). Mann–Whitney test * p < 0.05 IRI T30 vs. T0, T30 and sham. f Clone frequency analysis of Pax2+AQP2− cells in Pax2/Confetti mice at day 0 (T0, n = 4), in age-matched controls (T30, n = 5), in sham-operated mice (sham; n = 5) and at day 30 after IRI (IRI T30, n = 5). Mann–Whitney test * p < 0.05 IRI T30 vs. T0, T30, and sham. g Percentage of Pax2+AQP2− cells in Pax2/Confetti mice at day 0 (T0, n = 4) and 30 after IRI (IRI T30, n = 5). h Percentage of Pax2+AQP2− TECs vs. AQP2− TECs in Pax2/Confetti mice at day 0 (T0, n = 4), in age-matched controls (T30, n = 5), and at day 30 after IRI (IRI T30, n = 5). Mann–Whitney test * p < 0.05 IRI T30 vs. T0 and T30. i Percentage of lost Pax2+AQP2− TECs ( n = 5) and Pax8+AQP2− TECs ( n = 5) at IRI T30 vs. T0. Mann–Whitney test * p < 0.05. j Percentage of Pax2+AQP2− ( n = 5) and Pax8+AQP2− TECs ( n = 5) that generated clones at IRI T30 vs. T0. Mann–Whitney test * p < 0.05. k Percentage of Pax2+AQP2− ( n = 5) and Pax8+AQP2− TECs ( n = 5) at IRI T30 vs. T0. l GFR in Pax2/Confetti mice after nephrotoxic AKI ( n = 8) normalized on the GFR at baseline and on healthy mice ( n = 5). One-way ANOVA post-hoc Tukey. m Percentage of lost Pax2+AQP2− TECs ( n = 6) in Pax2/Confetti and of lost Pax8+AQP2− ( n = 4) in Pax8/Confetti mice at day 30 after nephrotoxic AKI vs. T0. Mann–Whitney test * p < 0.05. n Percentage of Pax2+AQP2− ( n = 6) and Pax8+AQP2− TECs ( n = 4) that generated clones at day 30 after nephrotoxic AKI vs. T0. Mann–Whitney test * p < 0.05. o Percentage of Pax2+AQP2− ( n = 6) and Pax8+AQP2− TECs ( n = 4) at day 30 after nephrotoxic AKI vs. T0. Data are mean ± SEM. Pax2+ = Pax2 lineage-positive cells, Pax8+ = Pax8 lineage-positive cells. For calculation of figures i – k , m – o see Methods section and representative calculations in Pax2/Confetti mice in Supplementary Methods

Article Snippet: The remaining part of the cells were incubated with anti-AQP2 pAb (bs-4611R, dilution 1:20, Bioss Inc.) followed by anti-rabbit MicroBeads (Miltenyi Biotec S.r.l.), then passed through MS columns (Miltenyi Biotec S.r.l.) accordingly with the manufacturer’s protocol and collecting the first flow-through containing unlabeled cells in order to perform a magnetic cell depletion of AQP2+ cells.

Techniques: Clone Assay, Staining, MANN-WHITNEY, Generated

Journal: Nature Communications

Article Title: Endocycle-related tubular cell hypertrophy and progenitor proliferation recover renal function after acute kidney injury

doi: 10.1038/s41467-018-03753-4

Figure Lengend Snippet: Pax2 lineage-positive cells regenerate long tubule segments. a Representative image of a kidney section showing single-colored clones in S1–S2 segments of proximal tubule as demonstrated by staining with anti-megalin antibody (white) in Pax2/Confetti mice at day 30 after IRI ( n = 5). Scale bar 20 µm. b , c Representative images of a kidney section showing single-colored clones in S3 segment of proximal tubule as demonstrated by staining with anti-AQP1 antibody (white) in OSOM of Pax2/Confetti mice at day 30 after IRI ( n = 5). Scale bars 20 µm. d Representative image of a kidney section showing single-colored clones in thick ascending limb as demonstrated by staining with anti-THP antibody (white) in Pax2/Confetti mice at day 30 after IRI ( n = 5). Scale bar 20 µm. e Frequency of Pax2+ clones in S1–S2 segment of proximal tubules (Pax2+ Megalin+ clones, n = 5), in S3 segment of proximal tubules (Pax2+AQP1+ clones, n = 7), in thick ascending limbs (TAL, Pax2+THP+ clones, n = 7) and in the collecting ducts (Pax2+AQP2+ clones, n = 7) at day 30 after IRI. Mann–Whitney test. ** p < 0.01 S3 vs. S1–S2 and collecting ducts, * p < 0.05 S3 vs. TAL and TAL vs. collecting ducts. f Percentage of clonogenic cells per field in 2D vs. 3D analysis in Pax2/Confetti and in Pax8/Confetti mice at day 30 after IRI vs. T0. ( n = 5 Pax2/Confetti mice and n = 5 Pax8/Confetti mice). Mann–Whitney test NS. g – i 3D reconstruction of single-colored clones in Pax2/Confetti mice at day 30 after IRI ( n = 5). DAPI counterstains nuclei (white). Scale bars 50 µm. Data are mean ± SEM. Pax2+ = Pax2 lineage-positive cells, Pax8+ = Pax8 lineage-positive cells

Article Snippet: The remaining part of the cells were incubated with anti-AQP2 pAb (bs-4611R, dilution 1:20, Bioss Inc.) followed by anti-rabbit MicroBeads (Miltenyi Biotec S.r.l.), then passed through MS columns (Miltenyi Biotec S.r.l.) accordingly with the manufacturer’s protocol and collecting the first flow-through containing unlabeled cells in order to perform a magnetic cell depletion of AQP2+ cells.

Techniques: Clone Assay, Staining, MANN-WHITNEY

Journal: Nature Communications

Article Title: Endocycle-related tubular cell hypertrophy and progenitor proliferation recover renal function after acute kidney injury

doi: 10.1038/s41467-018-03753-4

Figure Lengend Snippet: Pax2 lineage-positive cells proliferate, while other TEC endocycle and are persistently lost after AKI. a Schematic procedure. b – j FACS analysis shows mCherry+ and mVenus+ cells in total renal cells of Pax8/FUCCI2aR ( b , d ) and Pax2/FUCCI2aR mice ( c , d ) at T0, of Pax8/FUCCI2aR ( e , g ) and Pax2/FUCCI2aR mice ( f , g ) at IRI T2 and of Pax8/FUCCI2aR ( h , j ) and Pax2/FUCCI2aR mice ( i , j ) at IRI T30. A representative experiment out of 4 is shown. k Number of total FUCCI2aR cells in Pax8/FUCCI2aR and Pax2/FUCCI2aR mice at T0, IRI T2, IRI T30 ( n = 4 in each group). Mann–Whitney test * p < 0.05. l Number of new FUCCI2aR cells in Pax8/FUCCI2aR and Pax2/FUCCI2aR mice at IRI T30 in comparison to IRI T2 ( n = 4 in each group). Mann–Whitney test NS. m Percentage of Pax2/FUCCI2aR over Pax8/FUCCI2aR total cells at T0, IRI T2, IRI T30 ( n = 4 in each group). Mann–Whitney test * p < 0.05. n Number of total mVenus+ cells mCherry+mVenus+ cells in Pax8/FUCCI2aR and Pax2/FUCCI2aR mice at T0, IRI T2, IRI T30 ( n = 4 in each group). Mann–Whitney test NS. o , p Cell cycle distribution of mCherry+, mVenus+, and mCherry+mVenus+ cells in Pax8/FUCCI2aR ( o ) and Pax2/FUCCI2aR mice ( p ) at IRI T30. A representative experiment out of 4 is shown. q Percentage of endocycling cells in Pax2/FUCCI2aR (black column) and Pax8/FUCCI2aR mice (light gray column) at IRI T30 ( n = 4 in each group). Mann–Whitney test * p < 0.05. r mCherry+ cells (red) and p-H3+ cells (blue) in LTA+ tubules in the cortex (white) of Pax8/FUCCI2aR mice at IRI T30 ( n = 5). Arrows show mCherry+p-H3+ cells. r′ Detail of r . s mCherry+ cells (red) and mVenus+ cells (green) in LTA+ tubules in the cortex (white) of Pax8/FUCCI2aR mice at IRI T30 ( n = 5). Arrow shows mVenus+ cell. t Percentage of endocycling cells (mCherry+p-H3+ cells) and cycling cells (mVenus+ cells) in the cortex of Pax8/FUCCI2aR mice at IRI T30 ( n = 5). Mann–Whitney test * p < 0.05. u Percentage of endocycling (mCherry+p-H3+ cells) and cycling (mVenus+ cells) AQP2− cells in the cortex and in OSOM of Pax8/FUCCI2aR mice at IRI T30 ( n = 5). Mann–Whitney test * p < 0.05. v Measurement of the cell surface area of mCherry+p-H3+ cells and of mCherry+p-H3− cells after staining with Phalloidin (white) with Image J software in Pax8/FUCCI2aR mice at IRI T30 ( n = 4). w Cell surface area of mCherry+p-H3+ cells and of mCherry+p-H3− cells in Pax8/FUCCI2aR mice at IRI T30. n = at least 20 cells for each mouse ( n = 4), Mann–Whitney test *** p < 0.001. Data are mean ± SEM. Scale bars 20 µm. T0 = day 0, IRI T2 = day 2 after IRI, IRI T30 = day 30 after IRI

Article Snippet: The remaining part of the cells were incubated with anti-AQP2 pAb (bs-4611R, dilution 1:20, Bioss Inc.) followed by anti-rabbit MicroBeads (Miltenyi Biotec S.r.l.), then passed through MS columns (Miltenyi Biotec S.r.l.) accordingly with the manufacturer’s protocol and collecting the first flow-through containing unlabeled cells in order to perform a magnetic cell depletion of AQP2+ cells.

Techniques: MANN-WHITNEY, Staining, Software