CPPED1 Antibody

What is CPPED1 and what is its primary biological function?

CPPED1 is a serine/threonine protein phosphatase that plays an inhibitory role in glucose metabolism. Recent research has demonstrated that CPPED1 dephosphorylates AKT1 of the PI3K-AKT signaling pathway, a critical regulator of cellular metabolism . Studies in adipose tissue have shown that CPPED1 expression decreases after weight reduction, suggesting its involvement in metabolic adaptation . Functionally, CPPED1 appears to negatively regulate glucose uptake in adipocytes, with its knockdown improving insulin-stimulated glucose metabolism .

What experimental applications are suitable for CPPED1 antibodies?

CPPED1 antibodies have demonstrated utility in multiple experimental applications:

For IHC applications, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may serve as an alternative . These antibodies have been confirmed to show reactivity with human samples .

What are the expected molecular characteristics of CPPED1?

CPPED1 has a calculated molecular weight of 36 kDa (314 amino acids), though the observed molecular weight in experimental conditions typically ranges between 28-35 kDa . Multiple isoforms have been reported, including variants with molecular weights of approximately 20 kDa and 28-35 kDa . This variation should be considered when interpreting Western blot results, as band patterns may differ depending on tissue type and experimental conditions.

How should CPPED1 antibodies be stored and handled?

For optimal antibody performance, CPPED1 antibodies should be stored at -20°C in PBS buffer containing 0.02% sodium azide and 50% glycerol at pH 7.3 . Under these conditions, antibodies remain stable for one year after shipment. Notably, aliquoting is generally unnecessary for -20°C storage. Some antibody preparations may contain 0.1% BSA in smaller (20 μL) sizes . Always follow manufacturer guidelines for specific storage recommendations to maintain antibody integrity and performance.

What protocols are recommended for RNA interference of CPPED1?

For effective CPPED1 knockdown, RNA interference using small interfering RNA (siRNA) has been successfully employed. Published protocols have utilized ON-TARGETplus SMARTpool siRNA with four target sequences:

• AGAAAAUUGUUCACCGAUA

• UAAAUGCACUAAUGCGAAA

• CGGAGGACCUGAAGCGAGU

• CCUUUAAAAUGGAGCGAAU

For adipocyte models, transfection of 50 nmol/L siRNA using HiPerFect transfection reagent has produced effective knockdown, with verification by both RT-qPCR and Western blot at 48 hours post-transfection . When designing CPPED1 knockdown experiments, including appropriate scrambled siRNA controls is essential for result validation.

How does CPPED1 knockdown affect glucose metabolism pathways?

CPPED1 knockdown produces significant changes in glucose metabolism pathways, particularly in adipocytes. Experimental evidence demonstrates that siRNA-mediated reduction of CPPED1 leads to:

• Increased mRNA expression of adiponectin, adiponectin receptor 1, and GLUT4

• Decreased mRNA expression of GLUT1 and leptin (LEP)

• Enhanced insulin-stimulated glucose uptake by +74% compared to control cells

• Time-dependent increases in adiponectin protein expression (+32% at 96h post-treatment)

• Tendency toward increased high-molecular-weight adiponectin secretion

Notably, treatment with wortmannin (a PI3K inhibitor) abolishes the enhanced insulin-stimulated glucose uptake in both control and CPPED1 knockdown cells, suggesting this effect operates through the PI3K signaling pathway .

What methods are recommended for assessing CPPED1 expression in tissue samples?

Multiple approaches have been validated for detecting CPPED1 expression in tissue samples:

For immunohistochemical detection, researchers have successfully used rabbit anti-human CPPED1 antibody (HPA040938, 1:250 dilution) with the two-step Envision kit (K5007) for visualization . When analyzing expression patterns, consider that CPPED1 levels have been shown to decrease in subcutaneous adipose tissue following weight loss .

How can researchers troubleshoot specificity issues with CPPED1 antibodies?

When encountering specificity concerns with CPPED1 antibodies, consider these troubleshooting approaches:

• Verify antibody specificity through siRNA knockdown experiments, confirming reduced signal corresponds with reduced CPPED1 expression

• Optimize antigen retrieval methods for IHC applications (TE buffer pH 9.0 or alternatively citrate buffer pH 6.0)

• Adjust antibody dilutions based on sample type and application (refer to recommended ranges in section 1.2)

• Include appropriate negative controls in all experiments

• When possible, confirm results with multiple antibody clones or detection methods

For Western blot applications, special attention should be paid to the multiple isoforms of CPPED1, which may appear at different molecular weights (20 kDa and 28-35 kDa) .

What cell models are appropriate for studying CPPED1 function?

The Simpson-Golabi-Behmel syndrome (SGBS) cell strain has been successfully employed for functional studies of CPPED1 in adipocytes . These cells provide a reliable model for adipocyte differentiation and metabolic studies. For CPPED1 functional experiments, mature adipocytes (day 14 of differentiation) maintained in Dulbecco's modified Eagle's medium/Ham's F12 nutrient mixture (1:1) supplemented with 33 μmol/L biotin, 17 μmol/L pantothenate, 10 μg/mL transferrin, and 20 nmol/L insulin have shown appropriate responses to siRNA treatment .

Additionally, SMMC-7721 cells have demonstrated positive detection of CPPED1 in Western blot applications , making them potentially suitable for certain expression studies.

What methodologies are recommended for assessing glucose uptake after CPPED1 manipulation?

To effectively measure changes in glucose uptake following CPPED1 manipulation, researchers have employed insulin-stimulated glucose uptake assays. While specific protocols aren't detailed in the search results, standard approaches involve:

• Treating cells with or without CPPED1 siRNA

• Starving cells of serum and glucose

• Stimulating with insulin (or vehicle control)

• Measuring uptake of labeled glucose (typically radiolabeled or fluorescently labeled)

• Including pathway inhibitors (e.g., wortmannin) to determine mechanism specificity

This approach has demonstrated that CPPED1 knockdown increases insulin-stimulated glucose uptake by approximately 74% compared to control cells, an effect that is abolished by wortmannin treatment .

How should researchers design experiments to examine CPPED1's role in metabolic disorders?

When investigating CPPED1's involvement in metabolic disorders, researchers should consider several experimental design elements:

• Use appropriate metabolic disease models (e.g., diet-induced obesity, insulin resistance)

• Assess CPPED1 expression changes in relevant tissues (adipose tissue shows decreased CPPED1 after weight reduction )

• Include pathway analysis components (AKT phosphorylation status, PI3K activity)

• Measure functional outcomes (glucose uptake, insulin sensitivity)

• Consider examining CPPED1 in conjunction with established metabolic markers (adiponectin, GLUT4)

Given CPPED1's role in glucose metabolism regulation, examining its relationship to metabolic markers and insulin signaling pathways will provide insights into its potential significance in metabolic disorders.

What techniques are available for studying CPPED1 protein interactions?

While the search results don't specifically address protein interaction studies for CPPED1, several standard approaches would be appropriate based on CPPED1's known functions:

• Co-immunoprecipitation to identify protein binding partners

• Proximity ligation assays to visualize protein interactions in situ

• Phosphatase activity assays to measure CPPED1's enzymatic function

• Mass spectrometry-based interactome analysis

• Yeast two-hybrid screening to identify novel interaction partners

Given CPPED1's role in dephosphorylating AKT1 , techniques that specifically examine enzyme-substrate interactions would be particularly valuable.

How can researchers assess CPPED1's tissue-specific functions?

To investigate tissue-specific functions of CPPED1, researchers should consider employing:

• Tissue-specific knockdown or knockout models

• Comparative expression analysis across multiple tissues

• Examination of tissue-specific regulatory elements controlling CPPED1 expression

• Analysis of tissue-specific protein interactions and substrates

• Functional assays tailored to specific tissue types (e.g., glucose uptake in adipocytes, other relevant functions in other tissues)

Evidence already suggests tissue-specific differences in CPPED1 function, as its relationship with AKT1 phosphorylation appears to differ between adipose tissue and placenta .

What are the emerging research directions for CPPED1 in metabolic regulation?

Based on current research findings, future studies on CPPED1 might productively focus on:

• Further elucidating the molecular mechanisms of CPPED1's inhibitory effect on glucose uptake

• Exploring potential therapeutic applications targeting CPPED1 for metabolic disorders

• Investigating CPPED1's role in other PI3K-AKT-regulated processes beyond glucose metabolism

• Examining CPPED1 expression and function in various pathological states

• Identifying additional substrates for CPPED1's phosphatase activity

As CPPED1 has been described as "a novel molecule involved in AT biology" , many aspects of its function and regulation remain to be fully characterized, presenting numerous opportunities for innovative research.