anti ppp2c  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Protein Phosphatase 2A Subunit B55 Alpha is Required for Angiotensin Type 2 Receptor Elicited Natriuresis

doi: 10.1101/2025.04.07.647676

Figure Lengend Snippet: Sprague-Dawley rats were treated with control siRNA and PP2A B55α siRNA by renal interstitial (RI) infusion 48 hours prior to renal interstitial (RI) infusion of vehicle or compound 21 (C21; baseline, 20, 40, and 60 ng/kg/min, each dose for 30 minutes). Urine Na + excretion (U Na V) ( A ) and mean arterial blood pressures (MAP) ( B ) were measured. A. U Na V measurements with the following treatments: control siRNA right kidney (RT) RI infusion of vehicle, control siRNA left kidney (LT) RI infusion of C21, PP2A B55α siRNA right kidney (RT) RI infusion of vehicle, PP2A B55α siRNA left kidney (LT) RI infusion of C21. B. MAP measurements in rats in which control siRNA or PP2A B55α siRNA was infused in both kidneys and vehicle into right and C21 into left kidneys. Data are shown as mean±SE. Statistical significance was determined using a repeated measures analysis with an unstructured covariance matrix in the SAS PROC MIXED program. Overall comparison for all periods between Control siRNA Vehicle (RT) and Control siRNA C21 (LT); F=30.39, P<0.001. Overall comparison for all periods between Control siRNA C21 (LT) and PP2A B55α siRNA C21 (LT); F=22.83, P<0.001.

Article Snippet: Briefly, membranes after transfer and blocking were incubated overnight at 4°C with PP2A B subunit antibodies (Cell Signaling, cat. no. 2290; 1:1,000) in 5% BSA in TBST .

Techniques: Control, Comparison

Journal: bioRxiv

Article Title: Protein Phosphatase 2A Subunit B55 Alpha is Required for Angiotensin Type 2 Receptor Elicited Natriuresis

doi: 10.1101/2025.04.07.647676

Figure Lengend Snippet: Proximity Ligation Assays were performed on kidney sections obtained from Wistar Kyoto rats after renal interstitial infusion of vehicle or compound 21 (C21) in vivo . AT 2 R-specific antibodies were used in combination with antibodies against PP2A B55α (A) , PP2A A (C) , and PP2A B56γ (E) . Panels A , C, and E show representative confocal images with discrete green dots marking interactions between the AT 2 R and respective PP2A subunits. Panels B , D, and F show plots for the numbers of discrete green dots counted in the RPTC intracellular space (between the outer and inner white lines) and apical brush border membranes (inside the inner white line) representing interactions between the AT 2 R and PP2A B55α (B) , AT 2 R and PP2A A (D) , AT 2 R and PP2A B56γ (F) . Autofluorescence (blue, DAPI channel) was used to identify RPTCs. Data are shown as mean±SE (n=6) and were analyzed using one-way ANOVA.

Article Snippet: Briefly, membranes after transfer and blocking were incubated overnight at 4°C with PP2A B subunit antibodies (Cell Signaling, cat. no. 2290; 1:1,000) in 5% BSA in TBST .

Techniques: Ligation, In Vivo

Journal: bioRxiv

Article Title: Protein Phosphatase 2A Subunit B55 Alpha is Required for Angiotensin Type 2 Receptor Elicited Natriuresis

doi: 10.1101/2025.04.07.647676

Figure Lengend Snippet: HA-tagged AT 2 R was overexpressed in HEK293 cells and cell homogenates prepared under denaturing conditions to effectively disrupt protein interactions. After detergent neutralization, immunoprecipitations with HA-antibodies bound to magnetic beads pulled down HA-tagged AT 2 R (HA-AT 2 R IP). The HA-AT 2 R IP was incubated with buffer only (No PP2A B55α) or purified PP2A B55α (5 μg). Samples were separated by SDS PAGE and immunoblotted with antibodies against PP2A B55α (top panel) and AT 2 R (bottom panel). Rat kidney cortex homogenate (40 μg total protein) prepared as described , and purified PP2A B55α (0.2 μg) were loaded as positive controls in right two lanes.

Article Snippet: Briefly, membranes after transfer and blocking were incubated overnight at 4°C with PP2A B subunit antibodies (Cell Signaling, cat. no. 2290; 1:1,000) in 5% BSA in TBST .

Techniques: Neutralization, Magnetic Beads, Incubation, Purification, SDS Page

Journal: bioRxiv

Article Title: Protein Phosphatase 2A Subunit B55 Alpha is Required for Angiotensin Type 2 Receptor Elicited Natriuresis

doi: 10.1101/2025.04.07.647676

Figure Lengend Snippet: A. Representative confocal images depict renal proximal and distal tubule staining of PP2A B55α in control siRNA and PP2A B55α siRNA treated rats 48 hours after renal interstitial (RI) infusion of siRNA (23 μg). Apical membrane staining with phalloidin 750 marks proximal tubules. B . Quantification of PP2A B55α fluorescence intensity (RFU) in renal proximal and distal tubule cells of control siRNA and PP2A B55α siRNA-treated kidneys. C. Representative confocal images of control siRNA and PP2A B55α siRNA-treated kidneys 48 hours before RI infusion of vehicle or compound 21 (C21). D. and E. Quantifications of renal proximal tubule cell apical membrane (D) and total (E) PP2A B55α fluorescence intensity (RFU). Data are shown as mean±SE (n=6) and were analyzed using one-way ANOVA.

Article Snippet: Briefly, membranes after transfer and blocking were incubated overnight at 4°C with PP2A B subunit antibodies (Cell Signaling, cat. no. 2290; 1:1,000) in 5% BSA in TBST .

Techniques: Staining, Control, Membrane, Fluorescence

Journal: bioRxiv

Article Title: Protein Phosphatase 2A Subunit B55 Alpha is Required for Angiotensin Type 2 Receptor Elicited Natriuresis

doi: 10.1101/2025.04.07.647676

Figure Lengend Snippet: Sprague-Dawley rats were treated with control siRNA and PP2A B55α siRNA by renal interstitial (RI) infusion 48 hours prior to RI infusion of vehicle or compound 21 (C21) and kidney sections analyzed by confocal microscopy analysis. Representative confocal images show proximal tubule staining for AT 2 R (A) , NHE-3 (D) , and p-Src (G) . Quantification of RPTC apical brush border membrane AT 2 R (B) and NHE-3 (E) , and total RPTC AT 2 R (C) , NHE-3 (F) , p-Src (H) and c-Src (I) fluorescence intensity (RFU). Data are shown as mean±SE (n=6) and were analyzed using one-way ANOVA.

Article Snippet: Briefly, membranes after transfer and blocking were incubated overnight at 4°C with PP2A B subunit antibodies (Cell Signaling, cat. no. 2290; 1:1,000) in 5% BSA in TBST .

Techniques: Control, Confocal Microscopy, Staining, Membrane, Fluorescence

Journal: bioRxiv

Article Title: Protein Phosphatase 2A Subunit B55 Alpha is Required for Angiotensin Type 2 Receptor Elicited Natriuresis

doi: 10.1101/2025.04.07.647676

Figure Lengend Snippet: Sprague-Dawley rats were treated with control siRNA and PP2A B55α siRNA by renal interstitial (RI) infusion 48 hours prior to RI infusion of vehicle or compound 21 (C21) and kidney sections analyzed by confocal microscopy analysis. Kidney sections were co-stained with AT 2 R and LAMP1 antibodies and representative images of renal proximal tubule cells are shown for each condition and each antibody separately and together (merged) as labeled. Squares outlined in images at 1X were magnified (4X) to better show co-localization of AT 2 R with LAMP1.

Article Snippet: Briefly, membranes after transfer and blocking were incubated overnight at 4°C with PP2A B subunit antibodies (Cell Signaling, cat. no. 2290; 1:1,000) in 5% BSA in TBST .

Techniques: Control, Confocal Microscopy, Staining, Labeling

Journal: bioRxiv

Article Title: Protein Phosphatase 2A Subunit B55 Alpha is Required for Angiotensin Type 2 Receptor Elicited Natriuresis

doi: 10.1101/2025.04.07.647676

Figure Lengend Snippet: Under control siRNA/vehicle (VEH)-treated conditions, the AT 2 R recycles between the early endosomal (EE) compartment and the cell surface (apical brush border membranes) at a slow rate keeping AT 2 R at the cell surface low. A portion of the recycling AT 2 R in early endosomes traffics to late endosomes (LE) and lysosomes (LYS). AT 2 R agonist stimulation (C21) leads to increased AT 2 R binding to PP2A B55αC and AT 2 R/PP2A B55αC in apical brush border membranes, most likely due to a slow-down in AT 2 R internalization and/or promotion of AT 2 R recycling, allowing increased agonist binding and AT 2 R signaling to promote retrieval/internalization of the sodium transporters NHE3 and NKA and src phosphorylation and increase natriuresis. A smaller portion of the AT 2 R traffics to late endosomes during this phase. When B55α is knocked down, AT 2 R trafficking and signaling are disrupted and a large portion of the AT 2 R accumulates in lysosomes. The model was created using BioRender software.

Article Snippet: Briefly, membranes after transfer and blocking were incubated overnight at 4°C with PP2A B subunit antibodies (Cell Signaling, cat. no. 2290; 1:1,000) in 5% BSA in TBST .

Techniques: Control, Binding Assay, Software