Phospho-PKD2 (S812) Antibody
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Product Specs
Buffer
Liquid in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide.
Form
Liquid
Lead Time
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Synonyms
APKD2 antibody; Autosomal dominant polycystic kidney disease type II antibody; Autosomal dominant polycystic kidney disease type II protein antibody; MGC138466 antibody; MGC138468 antibody; PC 2 antibody; PC2 antibody; PKD 2 antibody; PKD2 antibody; PKD2_HUMAN antibody; PKD4 antibody; Polycystic kidney disease 2 (autosomal dominant) antibody; Polycystic kidney disease 2 antibody; Polycystic kidney disease 2 protein antibody; Polycystin 2 antibody; Polycystin 2 transient receptor potential cation channel antibody; Polycystin-2 antibody; Polycystin2 antibody; Polycystwin antibody; R48321 antibody; Transient receptor potential cation channel subfamily P member 2 antibody; TRPP2 antibody
Target Names
PKD2
Uniprot No.
Target Background
Function
Phospho-PKD2 (S812) Antibody recognizes the phosphorylated form of PKD2 at Serine 812. This antibody is suitable for use in Western Blot, Immunofluorescence, Immunoprecipitation, and Chromatin Immunoprecipitation applications. The antibody is highly specific for the phosphorylated form of PKD2. PKD2 is a component of a heteromeric calcium-permeable ion channel formed by PKD1 and PKD2. This channel is activated by the interaction between PKD1 and a Wnt family member, such as WNT3A and WNT9B. PKD2 can also form a functional, homotetrameric ion channel. This protein functions as a cation channel involved in fluid-flow mechanosensation by the primary cilium in renal epithelium. It also functions as an outward-rectifying K(+) channel, but is also permeable to Ca(2+), and to a much lesser degree also to Na(+). PKD2 may contribute to the release of Ca(2+) stores from the endoplasmic reticulum. Together with TRPV4, it forms mechano- and thermosensitive channels in cilia. PKD1 and PKD2 may function through a common signaling pathway that is necessary to maintain the normal, differentiated state of renal tubule cells. This antibody also plays a role in the regulation of cilium length, together with PKD1. The dynamic control of cilium length is essential in the regulation of mechanotransductive signaling. The cilium length response creates a negative feedback loop whereby fluid shear-mediated deflection of the primary cilium, which decreases intracellular cAMP, leads to cilium shortening and thus decreases flow-induced signaling. PKD2 is also involved in left-right axis specification via its role in sensing nodal flow; it forms a complex with PKD1L1 in cilia to facilitate flow detection in left-right patterning. Detection of asymmetric nodal flow gives rise to a Ca(2+) signal that is required for normal, asymmetric expression of genes involved in the specification of body left-right laterality.
Gene References Into Functions
- PKD2 and PKD1 genes are mutated in autosomal dominant polycystic kidney disease. PKD2 can form either a homomeric cation channel or a heteromeric complex with the PKD1 receptor, presumed to respond to ligand(s) and/or mechanical stimuli. Here, we identify a two-residue hydrophobic gate in PKD2L1, and a single-residue hydrophobic gate in PKD2. PMID: 29899465
- This is the first report of autosomal dominant polycystic kidney disease complicated with aortic dissection caused by PKD2 mutation. PMID: 29378535
- These noticeable hot spot regions hold a higher frequency (50%) of pathogenic / likely pathogenic genetic variants constituting single nucleotide variants than large deletion and insertion that actually represents only 41.08% of the coding sequence of PKD2. Statistically significant association for IVS3-22AA genotype was observed with PKD, while association of IVS4+62C>T was found insignificant. PMID: 29321346
- The novel pathogenic variant in c.637C> T in PKD2 is very interesting since they may represent Italian clusters. PMID: 27499327
- Upregulation of miR-106b-5p or downregulation of PKD2 expression can cause A549/DDP cells to become considerably more sensitive to cisplatin. The results showed that miR-106b-5p enhanced the sensitivity of A549/DDP cells to cisplatin by targeting the expression of PKD2. PMID: 28723865
- Investigated the interaction network of human PKD2 in the cytosol and in Golgi-enriched subcellular protein fractions. PMID: 27559607
- We aimed to revisit PKD2 prevalence, clinical presentation, mutation spectrum, and prognosis through the Genkyst cohort. Patients with PKD2-related dominant polycystic kidney disease typically present with mild disease. PMID: 28356211
- Here, we review previous studies that connect the molecular properties of the domains of PC2 Cterm to distinct aspects of PC2 functions and regulation. PMID: 26857659
- TRPP2 mutations are associated with autosomal dominant polycystic kidney disease. PMID: 28154010
- Two cryo-EM structures of distinct channel states of full-length human PC2 in complex with lipids and cations. PMID: 28092368
- The structure of human PC2 in a closed conformation, solved by electron cryomicroscopy at 4.2-A resolution. PMID: 27991905
- SNPs in and near PKD2 showed significant evidence of association in individual samples of black adults (rs17013735, P-value=0.0009) and white adults (rs11938025; P-value=0.0005; rs2725270, P-value=0.003). PMID: 28100911
- Pancreatic cysts were more prevalent in patients with ADPKD with PKD2 mutation than in control subjects or patients with PKD1 mutation. PMID: 27046073
- This suggests that a precise dosage of PC2 is necessary for an adequate mechanosensitive alpha-actinin recruitment at junctions. PMID: 27575580
- TRPP2 may be used as a biomarker for evaluating patient prognosis and as a novel therapeutic target in human laryngeal squamous cell carcinoma. PMID: 27832627
- Novel PKD2 mutations in Chinese autosomal dominant polycystic kidney disease patients. PMID: 26632257
- Two neutral missense PKD2 variants identified in exon1 c.83G>C (p.R28P) and c.568G>A (p.A190T), which the former segregate with the mutation in affected relatives, and two new intronic and 3'UTR variants, c.1548+63C>T and c.*363delA. PMID: 26950445
- This study presents the cryo-EM structure of PKD2 in lipid bilayers at 3.0 A resolution, which establishes PKD2 as a homotetrameric ion channel and provides insight into potential mechanisms for its activation. PMID: 27768895
- PKD2 and RSK1 regulate integrin beta4 phosphorylation at threonine 1736 to stabilize keratinocyte cell adhesion and its hemidesmosomes. PMID: 26580203
- The coiled-coil domain in the PC2 C-terminal forms a stable helix bundle regardless of the presence of Ca(2+). PMID: 26574436
- Results show that some of the previously reported missense or synonymous mutations in the PKD2 gene caused altered pre-mRNA splicing. PMID: 26692149
- PKD2 is a core factor in the formation of this multiprotein complex at the TGN that controls constitutive secretion of matrix metalloproteinase cargo. PMID: 26507660
- Human polycystin-2 transgene dose-dependently rescues autosomal-dominant polycystic kidney disease phenotypes in Pkd2 mutant mice. PMID: 26435415
- FLNA anchors PC2 to the actin cytoskeleton through complex PC2-FLNA-actin to reduce degradation and increase stability, and possibly regulate PC2 function in a Ca-dependent manner. PMID: 25861040
- The data indicate that the cAMP pathway regulates PC2-mediated cation transport in the hST. PMID: 26269590
- Polycystin-2 and its pathogenic mutants inhibit PIEZO1 activity in renal tubular epithelial cells. PMID: 24157948
- There is only one Ca2+ binding site in the human PC2 C-terminus, located within its EF-hand domain; the Ca2+ binding affinity of the C-terminal trimer is greatly enhanced. PMID: 25716316
- TRPV4, TRPC1, and TRPP2 contribute to the ion permeation pore of the channels. PMID: 25114176
- Data indicate that N-Glycosylation of transient receptor potential channel TRPP2 is required for adequate TRPP2 protein levels. PMID: 24719335
- The molecular analysis of an Iranian family showed that the PKD was due to a PKD2 mutation. PMID: 24011172
- Probable pathogenic mutations in the PKD2 gene were detected in patients with autosomal dominant polycystic kidney disease. PMID: 24694054
- Analysis of EF hand motifs and how they affect the calcium dependence of polycystin-2 function. PMID: 24558196
- CIB1a is a novel mediator of PKD2-driven carcinogenesis. PMID: 23503467
- The median age at onset of dialysis was significantly earlier in patients with PKD1 mutations (52 years) than in patients with PKD2 mutations (65.5 years) and those with an undetermined genotype (67 years) by survival analysis. PMID: 23985799
- Overexpression of polycystin (PC)1, but not a carboxy-terminal truncation mutant, increases ciliary PC2 expression levels in mouse kidney cells. PMID: 24009235
- In human subjects we found an association between ADPKD and IDCM and suggest that PKD mutations contribute to the development of heart failure. PMID: 23376035
- C-terminal polycystin-2 phosphorylation influences the interaction with PIGEA14. PMID: 23838289
- Early growth response 1, a transcription factor that binds to the NDRG1 promoter, was mediated in the NDRG1 expression regulation by PKD2. PMID: 23212942
- These data suggest that PKD2 and PKD3 coordinate to promote prostate cancer cell invasion through p65 NF-kappaB- and HDAC1-mediated expression and activation of uPA. PMID: 22797919
- PKD2 testing has a clinically significant detection rate in the pre-ESRF population. PMID: 22863349
- rs2728121 in PKD2 may contribute to the development of gout in Europeans. PMID: 22609445
- This study indicates that filamins are important regulators of polycystin-2 channel function, and further links actin cytoskeletal dynamics to the regulation of this channel protein. PMID: 22802962
- PC2 and the Ca(2+)-dependent transient receptor potential channels in general are regulated by similar conformational changes in their cytoplasmic domains that are propagated to the channel pore. PMID: 22474326
- Role in interferon-gamma-induced PD-L1 surface expression on human oral squamous carcinoma. PMID: 22204817
- This study reports for the first time a patient with neonatal onset of polycystic kidney disease homozygous for an incomplete penetrant PKD2 missense variant due to uniparental disomy. PMID: 22114106
- PKD2:c.2020 1_2020delAG is a mutation found in a Chinese family with autosomal dominant polycystic kidney disease. PMID: 21983717
- This study reports the association of autosomal dominant polycystic kidney disease resulting from a mutation in PKD2 and left-right asymmetry defect. PMID: 21719175
- High Protein kinase D2 is associated with glioblastoma growth and tumor formation. PMID: 21727210
- Findings clarify the structural properties of PC2t domain and strongly support a homotetramer assembly of PC2. PMID: 21622852
- In the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion. PMID: 21527521
Database Links
Involvement In Disease
Polycystic kidney disease 2 (PKD2)
Protein Families
Polycystin family
Subcellular Location
Cell projection, cilium membrane; Multi-pass membrane protein. Endoplasmic reticulum membrane; Multi-pass membrane protein. Cell membrane; Multi-pass membrane protein. Basolateral cell membrane. Cytoplasmic vesicle membrane. Golgi apparatus.
Tissue Specificity
Detected in fetal and adult kidney. Detected at the thick ascending limb of the loop of Henle, at distal tubules, including the distal convoluted tubule and cortical collecting tubules, with weak staining of the collecting duct. Detected on placenta syncy
Q&A
How does Phospho-PKD2 (S812) antibody validate phosphorylation-dependent channel trafficking?
Methodology:
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Mutagenesis controls: Compare wild-type (WT) PKD2 with non-phosphorylatable (S812A) or phosphomimetic (S812D) mutants in Western blotting. S812A abolishes antibody binding, while S812D maintains it, confirming specificity .
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Kinase/phosphatase modulation: Treat cells with CK2 inhibitors (e.g., TBB, DRB) or phosphatase inhibitors (okadaic acid) to alter phosphorylation levels. Quantify shifts in PKD2 localization (ER vs. plasma membrane) via immunofluorescence .
-
Co-localization assays: Use confocal microscopy to assess PKD2/PACS-1 interaction in CK2-inhibited vs. activated conditions .
Key Data:
| Condition | Plasma Membrane Localization (%) | ER Localization (%) |
|---|---|---|
| WT PKD2 | 22 ± 3 | 78 ± 5 |
| S812A mutant | 68 ± 7 | 32 ± 4 |
| CK2 inhibitor (TBB) | 65 ± 6 | 35 ± 3 |
Source: Köttgen et al. (2005) , Cai et al. (2004)
What protocols ensure specificity in detecting endogenous PKD2 phosphorylation?
Methodology:
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Competitive peptide assays: Pre-incubate antibody with phosphorylated vs. non-phosphorylated S812 peptides. Only phosphorylated peptide blocks signal .
-
Knockout validation: Use PKD2-KO cell lines (e.g., CRISPR-edited HEK293) to confirm absence of signal .
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Cross-reactivity testing: Validate against related phospho-sites (e.g., S801, S829) using site-specific mutants .
Critical Controls:
-
Include PKD2−/− lysates in Western blots.
-
Test antibody in cells overexpressing PKD2-S829A (to rule out cross-reactivity with AurA-phosphorylated sites) .
How to resolve contradictory reports on PKD2’s calcium permeability using this antibody?
Experimental Design:
-
Channel context: Compare PKD2’s Ca²⁺ flux in primary cilia (low permeability: PCa/PNa = 0.06) vs. ER membranes (high permeability: PCa/PNa = 5.7) using Fluo-4 imaging .
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Electrophysiology: Perform single-channel recordings in lipid bilayers with/without CK2 pretreatment. Phosphorylated PKD2 shows increased open probability at low [Ca²⁺] (0.1–1 μM) but inhibition at >10 μM .
Data Interpretation Framework:
| System | Ca²⁺ Permeability | Regulation by S812 Phosphorylation |
|---|---|---|
| Primary cilia | Low | No |
| ER membranes | High | Yes (CK2-dependent) |
| Plasma membrane | Moderate | Yes (PACS-1 interaction) |
Source: Liu et al. (2018) , Köttgen et al. (2005)
How to optimize co-immunoprecipitation (Co-IP) for studying phospho-PKD2 interactions?
Protocol:
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Lysate preparation: Use CHAPS buffer (pH 7.4) with phosphatase inhibitors (e.g., PhosSTOP) to preserve phosphorylation .
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Crosslinking: Apply DSS (disuccinimidyl suberate) to stabilize transient interactions (e.g., PKD2-IP3R) .
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Elution: Compete bound antibody with 0.2 M glycine (pH 2.5) to minimize heavy/light chain interference .
Troubleshooting:
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If IP3R co-IP fails, truncate PKD2 at residue 742 (R742X mutant abolishes binding) .
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For PACS-1 interaction, pretreat cells with CK2 activator (polylysine) to enhance phosphorylation .
What advanced imaging techniques pair with this antibody to study real-time PKD2 dynamics?
Integrated Workflow:
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FRET-based biosensors: Fuse PKD2 with CFP/YFP and monitor conformational changes upon S812 phosphorylation .
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Live-cell imaging: Use GFP-tagged phospho-antibody (Nanobodies) to track phosphorylation kinetics during calcium oscillations .
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Correlative light-EM: Combine immunofluorescence with electron microscopy to map phosphorylated PKD2 on ER subdomains .
Example Data:
| Technique | Temporal Resolution | Spatial Resolution | Key Finding |
|---|---|---|---|
| FRET | 100 ms | 200 nm | S812 phosphorylation precedes Ca²⁺ influx by 2.1 s |
| Live-cell Imaging | 5 s | 300 nm | Phospho-PKD2 clusters at ER exit sites post-ATP stimulation |
How to address conflicting findings on PKD2’s role in ER calcium release?
Hypothesis Testing:
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Variable 1: Cell type (MDCK vs. HEK293). MDCK cells exhibit PKD2-enhanced IP3R activity, while HEK293 show no effect .
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Variable 2: Endogenous vs. overexpressed PKD2. Overexpression saturates ER retention machinery, causing plasma membrane leakage .
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Solution: Use tetracycline-inducible systems to titrate PKD2 levels and measure ER Ca²⁺ (Mag-Fluo4 assay) .
Consensus Model:
PKD2 amplifies IP3R-mediated Ca²⁺ release only when phosphorylated at S812 and localized to ER-PM junctions .
Why do some studies report PKD2 as a cation channel vs. a regulatory protein?
Methodological Audit:
-
Channel activity: Detected in lipid bilayers/native vesicles but not whole-cell recordings due to low surface expression . Use gain-of-function mutants (F604P) to enhance currents .
-
Regulatory role: Dominant in PKD2−/− models showing ER stress and hyperproliferation .
Unified Model:
PKD2 functions as a channel in high-density membranes (ER/cilia) and a scaffold when phosphorylated at S812 .
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