PPP1R3B Antibody

What is PPP1R3B and what is its function in metabolism?

PPP1R3B (protein phosphatase 1, regulatory inhibitor subunit 3B) is a glycogen regulatory protein also known as GL that plays an established role in liver glycogen metabolism and plasma glucose homeostasis. The protein functions as a metabolic switch that influences hepatic energy storage mechanisms, affecting both glycogen synthesis and lipid accumulation pathways in the liver . PPP1R3B has been associated with multiple cardiometabolic traits through genome-wide association studies (GWAS), including fasting glucose levels, insulin levels, plasma lipids, and indicators of hepatic steatosis and liver damage . The protein has a calculated molecular weight of 33 kDa, which matches its observed size in experimental systems .

What applications are validated for PPP1R3B antibody in research?

PPP1R3B antibodies have been validated for several key laboratory applications. According to technical validation data, the 14190-1-AP antibody from Proteintech has been tested and confirmed for Western Blot (WB), Immunohistochemistry (IHC), and ELISA applications . The antibody shows specific reactivity with human and mouse samples, making it suitable for comparative studies across these species . Published literature demonstrates successful application in knockout/knockdown studies as well, providing further evidence for antibody specificity and utility in functional genomics research .

What are the optimal storage and handling conditions for PPP1R3B antibodies?

For maintaining antibody stability and performance, PPP1R3B antibodies should be stored at -20°C in appropriate buffer conditions. The 14190-1-AP antibody is supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . These storage conditions ensure antibody stability for up to one year after shipment when properly maintained. Aliquoting is generally unnecessary for -20°C storage, simplifying laboratory handling procedures. Smaller packaging sizes (20μl) may contain 0.1% BSA as a stabilizer . These conditions are optimized to maintain antibody performance across multiple freeze-thaw cycles.

What protocols are recommended for PPP1R3B antibody in Western blotting?

For Western blot applications, PPP1R3B antibodies should be used according to the following methodological guidelines:

Researchers should optimize the specific dilution based on their detection system and sample type, as antibody performance can be sample-dependent . Standard Western blot protocols should be followed, including appropriate blocking, washing, and exposure steps. The antibody has been validated to detect the expected 33 kDa band corresponding to PPP1R3B protein .

What methodology should be used for immunohistochemistry with PPP1R3B antibody?

For immunohistochemical detection of PPP1R3B, researchers should follow these methodological guidelines:

Successful immunohistochemical detection has been validated in human stomach cancer tissue . The antibody may be used to detect PPP1R3B in various tissue contexts, including liver where the protein is highly expressed. Researchers should perform careful titration experiments to determine optimal antibody concentration for their specific tissue samples and detection systems .

How can PPP1R3B transcript variants be quantified using qPCR?

For accurate quantification of PPP1R3B transcript variants by qPCR, researchers should use variant-specific primers:

For PPP1R3B transcript variant 1:

• Forward primer: 5'-CCT CGG GAC TTA TGA GCT GAA-3'

• Reverse primer: 5'-GAG CCA TGC AGT TGT ATC TGT ACT C-3'

• Probe: 5'-ATC TAG CCC CAT GAT GGC TGT GGA CAT-3'

For PPP1R3B transcript variant 2:

• Forward primer: 5'-CGG CCC AAA AGC CTG TT-3'

• Reverse primer: 5'-GAG CCA TGC AGT TGT ATC TGT ACT C-3'

• Probe: 5'-ATC TAG CCC CAT GAT GGC TGT GGA CAT-3'

These primer sets can be used to amplify the respective transcript variants from cDNA prepared from research samples. According to expression profiling studies, PPP1R3B transcript variant 2 shows expression in multiple tissues, including placenta, leukocytes, prostate, and spleen, while both variants are expressed at high levels in various melanoma cell lines .

How do PPP1R3B mutations impact T cell recognition in cancer immunotherapy?

Research has identified PPP1R3B as a potential target in cancer immunotherapy, particularly in the context of melanoma treatment. A case study demonstrated that a somatic mutation in PPP1R3B (a C to A transversion at position 527 bp, resulting in a substitution of histidine for proline at position 176) was recognized by tumor-infiltrating lymphocytes (TILs) used in adoptive cell therapy . This mutated PPP1R3B epitope represented the immunodominant target recognized by tumor-reactive T cells in a patient who experienced complete response with regression of liver tumor masses and sustained remission for over seven years .

The mechanism involves:

• Mutation-specific recognition by CD8+ T cells

• Presentation of the mutated epitope by HLA-A*01

• Persistence of mutation-specific T cells in circulation for years after treatment

Methodologically, researchers identified this interaction through cDNA library screening, where cells transfected with the mutant PPP1R3B induced IFN-γ secretion from patient-derived TILs . This finding highlights the importance of somatic mutations in generating neoantigens for cancer immunotherapy.

What are the metabolic consequences of altered PPP1R3B expression in liver tissue?

Altered PPP1R3B expression has significant and divergent effects on hepatic metabolism, as demonstrated in mouse models with either hepatocyte-specific deletion or overexpression of the gene. The metabolic phenotypes include:

These findings indicate that PPP1R3B functions as a metabolic switch that shifts hepatic energy storage between glycogen and lipid pathways . Importantly, both deletion and overexpression lead to elevated ALT levels, suggesting that tight regulation of PPP1R3B is necessary for normal liver function. These changes are particularly pronounced when mice are challenged with high-sucrose diets, suggesting an interaction between PPP1R3B function and dietary carbohydrate intake .

How do genetic variants in the PPP1R3B locus correlate with clinical outcomes in human populations?

Human genetic studies have identified several noncoding variants in the PPP1R3B locus that are associated with altered PPP1R3B expression and metabolic traits. Analysis of the Penn Medicine BioBank (PMBB) revealed that carriers of specific variants (rs4240624, rs4841132, and rs9987289, which are in strong linkage disequilibrium) displayed:

• Elevated plasma ALT levels, suggesting liver damage

• Decreased hepatic fat as estimated by CT attenuation

Conversely, carriers of putative loss-of-function (pLOF) PPP1R3B variants, though rare in the population, showed significantly higher CT-derived hepatic fat (β= 20.83; p= 0.0012) . These human genetic findings align with the phenotypes observed in mouse models, where PPP1R3B overexpression reduces hepatic fat accumulation while deletion promotes it.

These associations demonstrate the clinical relevance of PPP1R3B in human metabolism and liver health, suggesting that modulation of PPP1R3B activity could have therapeutic implications for metabolic disorders. The contrasting effects on liver fat and liver damage markers highlight the complex role of PPP1R3B in hepatic metabolism .

What mouse models are available for studying PPP1R3B function?

Several genetically engineered mouse models have been developed to study PPP1R3B function in vivo:

• Hepatocyte-specific deletion model (Ppp1r3bDhep): This model exhibits:

  • Dramatic reduction in glycogen synthase activity

  • Depletion of liver glycogen stores

  • Rapid fasting hypoglycemia

  • Increased hepatic triglyceride accumulation

  • Histological evidence of steatosis with positive PLIN2 staining

• Hepatocyte overexpression model (Ppp1r3bhepOE): This model demonstrates:

  • Increased liver glycogen content

  • Preserved blood glucose levels after prolonged fasting

  • Improved glucose disposal

  • Reduced hepatic triglycerides

  • Increased glycogen deposition visible with PAS staining

These models can be challenged with different dietary conditions, such as high-sucrose diet (HSD, 66% sucrose) to exacerbate metabolic phenotypes . The contrasting phenotypes between these models provide valuable insights into the role of PPP1R3B in regulating the balance between carbohydrate and lipid metabolism in the liver.

What HLA binding assays can be used to study PPP1R3B peptide recognition?

For researchers investigating the immunological properties of PPP1R3B peptides, particularly in the context of cancer immunotherapy, the following HLA binding assay methodology has been validated:

• Treatment of EBV-transformed B cells with acid to remove HLA-bound peptides

• Incubation for 24 hours at 4°C with:

  • A fluorescent reference peptide (Fl-A1 [YLEPAC(Fl)AKY])

  • Various concentrations of test peptides

• Flow cytometric analysis to calculate the percentage of inhibition of fluorescent peptide binding

This methodology has been used to test binding of wild-type and mutant PPP1R3B peptides to HLA molecules, specifically:

• PPP1R3B 172wt (YTDFPCQYVK)

• PPP1R3B 172mut (YTDFHCQYVK)

The assay provides quantitative data on peptide binding affinity, expressed as IC50 values, which are critical for understanding the immunological recognition of wild-type versus mutated PPP1R3B epitopes .

What emerging areas of PPP1R3B research show the most promise?

Based on current findings, several promising research directions for PPP1R3B include:

• Therapeutic targeting of PPP1R3B for metabolic disorders: As PPP1R3B overexpression improves postprandial glucose clearance from the blood, it represents a potential therapeutic target for type 2 diabetes and related metabolic disorders .

• PPP1R3B in cancer immunotherapy: The identification of mutated PPP1R3B as a target for tumor-infiltrating lymphocytes suggests potential applications in personalized cancer immunotherapy, particularly for identifying patient-specific neoantigens .

• PPP1R3B as a biomarker for liver disease risk: Given the associations between PPP1R3B variants and liver fat content/damage markers, further investigation of PPP1R3B as a biomarker for NAFLD risk and progression is warranted .

• Structure-function studies of PPP1R3B: Detailed investigation of how specific domains of PPP1R3B regulate the balance between glycogen synthesis and lipid accumulation could provide mechanistic insights for targeted interventions.