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- Table of Contents
Facts about Phosphoenolpyruvate carboxykinase [GTP], mitochondrial.
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Human | |
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Gene Name: | PCK2 |
Uniprot: | Q16822 |
Entrez: | 5106 |
Belongs to: |
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phosphoenolpyruvate carboxykinase [GTP] family |
EC 4.1.1.32; PE; PEPCK; PEPCK2PEP carboxykinase; PEPCK-M; phosphoenolpyruvate carboxykinase [GTP], mitochondrial; phosphoenolpyruvate carboxykinase 2 (mitochondrial); Phosphoenolpyruvate carboxylase; phosphopyruvate carboxylase
Mass (kDA):
70.699 kDA
Human | |
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Location: | 14q11.2-q12 |
Sequence: | 14; NC_000014.9 (24094311..24104125) |
Mitochondrion.
This article will give you a brief introduction to Steven Boster and his work on the PCK2 marker. We will also talk about the background, High-affinity primary antibodies, and RT-PCR. These three techniques are essential for research projects, and Boster offers many antibodies that are highly effective for various applications. You will also learn more about Boster Bio's PCK2 antibodies and what makes them a superior choice for your research needs.
The role of the PCK2 marker in the formation of bone is still unclear. It has been implicated in metabolic bone diseases and stem cell-based bone tissue engineering. The PCK2 gene has two variants, one of which lacks multiple 3' coding exons. In variant 2, the 3' terminal exon extends beyond the splice site used in variant 1. The encoded isoform (2) also has a distinct C-terminus and is shorter than variant 1.
After seven days of osteogenic differentiation, PCK2 overexpression cells exhibited increased expression of RUNX2 and ALP genes. These cells also displayed elevated gene expressions of osteogenic markers. Furthermore, cells overexpressing PCK2 showed enhanced extracellular matrix mineralization. However, the levels of PCK2 protein were decreased in cells with low PCK2 expression. Therefore, PCK2 overexpression is a potential tumor suppressor.
In addition, the PCK2 gene has a profound impact on RCC biology. The GSEA gene enrichment analysis using the TCGA database revealed that PCK2 has high correlations with three key basal metabolic processes in RCC. These are glucose metabolism, lipid metabolism, and glutathione metabolism. These processes are critical to normal physiological activities of the cells, and disrupting the balance leads to an unbalanced intracellular environment.
RCC cells overexpressing PCK2 exhibit increased sensitivity to sunitinib. This is thought to be due to methylation of the PCK2 promoter region. PCK2 mRNA expression correlates with the levels of various proteins involved in endoplasmic reticulum stress. The findings suggest that PCK2 promotes cell proliferation and promotes the expression of RCC cells. The overexpression of the PCK2 gene increases the expression of ER-stress sensors in RCC cells.
The results indicate that PCK2 phosphorylates NF-kB in the cytosol of Kupffer cells. This suggests that it may be a novel therapeutic target in the regulation of inflammatory responses by Kupffer cells. The study concludes that PCK2 promotes the phosphorylation of the AKT/MAPK pathway. If it works as a potential target in disease prevention, it could be used as a biomarker in clinical trials.
To make high-affinity primary antibodies against the human PCK2 protein, we used a peptide targeting the carboxy terminus of the human protein. The peptide is synthesized from the human PCK2 protein and is used as a marker for antibody production. After the antibody is produced, it is purified using protein A or peptide affinity chromatography. This protocol is ideal for the detection of phosphorylated PCK2 in a variety of biological samples.
The promoter region of PCK2 was amplified from human genomic DNA and cloned into a luciferase reporter vector (PGL3-basic). The promoter sequence of the PCK2 gene varies from -214 to -1285. The transient transfections of -214 and -808 promoters were performed in glutamine-deprived MCF7 cells to test the efficacy of the antigens.
The antigen is expressed in cancer cells and is regulated by amino acid limitation. After subcellular fractionation, protein expression was assessed using Western blot. To identify other cell types, we used a positive control for PEPCK-M, mitochondrial superoxide dismutase, and mitochondrial voltage-dependent anion channel protein. For loading control, we used Coomassie Blue.
RT-PCR with the PCK2 gene marker was used to determine if the phosphorylation of PCK2 is involved in cell proliferation. The PCK2 promoter region was amplified from human genomic DNA, and cloned into a luciferase reporter vector, PGL3-basic. To determine whether the phosphorylation of PCK2 is associated with cell proliferation, cells were transfected with ATF4 siRNA and incubated for eight hours in glutamine-free medium or with thapsigargin.
The expression of PCK2 increased in Kupffer cells, whereas it decreased in hepatocytes. This metabolic shift may relate to the increase in PCK2 in the body's cells following exposure to LPS. The LPS induced inflammatory responses in the human body due to an increase in the level of PCK2. Hence, it's important to identify the specific mechanisms underlying the inflammatory response.
RNA was extracted from liver samples, fixed overnight in 4% paraformaldehyde, and processed with hematoxylin and eosin. After processing, liver cells were stained for hepatic glycogen with a PAS staining kit. PEPCK activity was determined with a PEPCK activity kit purchased from the Nanjing Jian Cheng Bioengineering Institute.
RT-PCR with the PCK2 gene was also successful in detecting inflammatory reactions and identifying cancer cells. PCK2 is expressed in cancer cells, but it is undetectable in normal cells. The expression of this gene has been linked to the survival of PCa patients. Therefore, the detection of a cancer cell based on PCK2 expression levels requires a high-quality panel of tumors.
RT-PCR with the PCK2 gene was performed to determine if a cancer cell has the mutation. Total RNA was isolated using an UltraspecTM RNA isolation system. Two milligrams of RNA were used to synthesize cDNA. The cDNA was then analyzed using a High Capacity cDNA reverse transcription kit from Invitrogen. In the next step, cDNA was synthesized using the ACTB gene as an internal reference. The gene expression was calculated as the mean of three independent tests.
Another important application of PCK2 is in the investigation of cell proliferation. It is known to be involved in cell proliferation by modulating the protein phosphorylation of signaling pathways. Therefore, knockdown of this gene inhibits both ERK and AKT. Moreover, knockdown of this gene inhibits the activity of these two proteins, which in turn suppresses cell proliferation. In addition to this, PCK2 inhibits the expression of other genes and NF-kb.
PMID: 8645161 by Modaressi S., et al. Molecular cloning, sequencing and expression of the cDNA of the mitochondrial form of phosphoenolpyruvate carboxykinase from human liver.
PMID: 9657976 by Modaressi S., et al. Human mitochondrial phosphoenolpyruvate carboxykinase 2 gene. Structure, chromosomal localization and tissue-specific expression.