|Detection of Human/Mouse/Rat PTEN by Western Blot. Western blot shows lysates of mouse and rat brain tissue, A431 human epithelial carcinoma cell line, and MRC-5 human embryonic lung fibroblast cell line. PVDF membrane was probed with 0.1 µg/mL Rabbit Anti-Human/Mouse/Rat PTEN Antigen Affinity-purified Polyclonal Antibody (Catalog # AF847) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). For additional reference, recombinant human PTEN (5 ng) was included. A specific band for PTEN was detected at approximately 54 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 4.|
|PTEN in Human Liver. PTEN was detected in immersion fixed paraffin-embedded sections of human liver using Rabbit Anti-Human/Mouse/Rat PTEN Antigen Affinity-purified Polyclonal Antibody (Catalog # AF847) at 15 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Rabbit HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS005) and counterstained with hematoxylin (blue). Lower panel shows a lack of labeling if primary antibodies are omitted and tissue is stained only with secondary antibody followed by incubation with detection reagents. View our protocol for Chromogenic IHC Staining of Paraffin-embedded Tissue Sections.|
Intracellular Staining by Flow Cytometry
|Detection of PTEN in Human PBMC lymphocytes by Flow Cytometry. Human peripheral blood lymphocytes were stained with Rabbit Anti-Human/Mouse/Rat PTEN Antigen Affinity‑purified Polyclonal Antibody (Catalog # AF847, filled histogram) or control antibody (Catalog # AB‑105‑C, open histogram), followed by Phycoerythrin-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # F0110). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with saponin.|
|Detection of Human PTEN by Simple WesternTM. Simple Western lane view shows lysates of A431 human epithelial carcinoma cell line and HeLa human cervical epithelial carcinoma cell line, loaded at 0.2 mg/mL. A specific band was detected for PTEN at approximately 60 kDa (as indicated) using 1 µg/mL of Rabbit Anti-Human/Mouse/Rat PTEN Antigen Affinity-purified Polyclonal Antibody (Catalog # AF847). This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.|
The tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome 10), also known as MMAC1 (mutated in multiple advanced cancers 1), encodes a phosphatase that contains the catalytic signature motif (HCXXGXXRS/T) found in all members of the protein tyrosine phosphatase family. In vitro, the recombinant PTEN has both lipid phosphatase and protein phosphatase activities (1, 2). Interestingly, accumulating evidence has shown that the tumor suppressor activity of PTEN relies on its ability to dephosphorylate phosphatidylinositol (3, 4, 5)-triphosphate specifically at position 3 of the inositol ring (3). This activity reduces the levels of phosphatidylinositol (3, 4, 5)-triphosphate which is specifically produced from phosphatidylinositol (4, 5)-diphosphate by PI 3-kinase upon activation by a variety of stimuli. Therefore, PTEN antagonizes PI 3-kinase-induced downstream signaling events and cellular processes including cell growth, apoptosis and cell motility. In vivo, the importance of PTEN catalytic activity in its tumor suppressor functions is underscored by the fact that the majority of PTEN missense mutations detected in tumor specimens target the phosphatase domain and cause a loss in PTEN phosphatase activity (4).
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