PTEN KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, 11 bp deletion in exon 5 and 5 bp insertion in exon 5 and Insertion of the selection cassette in exon 5.
10q23del, BZS, DEC, GLM2, MGC11227, MHAM, MMAC1, MMAC1 phosphatase and tensin homolog deleted on chromosome 10, Mutated in multiple advanced cancers 1, PTEN1, PTEN_HUMAN, Phosphatase and Tensin Homolog, Phosphatase and tensin like protein, Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase PTEN
PTEN KO cell line available to order. KO validated by Western blot. Free of charge wild type control provided. Knockout achieved by using CRISPR/Cas9, 11 bp deletion in exon 5 and 5 bp insertion in exon 5 and Insertion of the selection cassette in exon 5.
Upon arrival, the vial should be stored in liquid nitrogen vapor phase and not at -80°C. Storage at -80°C may result in loss of viability.
1. Thaw the vial in 37°C water bath for approximately 1-2 minutes.
2. Transfer the cell suspension (0.8 mL) to a 15 mL/50 mL conical sterile polypropylene centrifuge tube containing 8.4 mL pre-warmed culture medium, wash vial with an additional 0.8 mL culture medium (total volume 10 mL) to collect remaining cells, and centrifuge at 201 x g (rcf) for 5 minutes at room temperature. 10 mL represents minimum recommended dilution. 20 mL represents maximum recommended dilution.
3. Resuspend the cell pellet in 5 mL pre-warmed culture medium and count using a haemocytometer or alternative cell counting method seed all remaining cells into a T25.
4. Incubate the culture at 37°C incubator with 5% CO2. Check the culture one day after revival and continue to check until 80% confluent. Media change can be given if needed.
5. Once confluent passage into an appropriate flask at a density of 2x104 cells/cm2. Seeding density is given as a guide only and should be scaled to align with individual lab schedules. Cultures should be monitored daily.
We will provide viable cells that proliferate on revival.
This product is subject to limited use licenses from The Broad Institute, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and patents please refer to our limited use license and patent pages.
The PTEN protein also known as phosphatase and tensin homolog is a phosphatase enzyme with a molecular mass of approximately 47 kDa. It acts mechanically by removing phosphate groups from phosphatidylinositol (345)-trisphosphate (PIP3) converting it to phosphatidylinositol (45)-bisphosphate. PTEN is ubiquitously expressed in various tissues with pronounced presence in the brain lung kidney and testis. This enzyme is an important regulator of cellular functions through its impact on signaling pathways.
PTEN plays important roles in cellular processes like apoptosis cell proliferation and migration. It negatively regulates the PI3K/AKT signaling pathway critical for cell survival and growth. PTEN is not part of a larger protein complex but interacts with various other proteins modulating its activity. It maintains cellular homeostasis by balancing growth-promoting signals with cell cycle arrest and apoptotic pathways.
PTEN is an important component in the PI3K/AKT and mTOR signaling pathways. These pathways regulate cell growth metabolism and survival and are interlinked with insulin signaling and cancer progression. PTEN's function directly interacts with proteins such as AKT and mTOR serving as a checkpoint that ensures controlled cellular proliferation. This positions PTEN as a tumor suppressor inhibiting uncontrolled cell growth via modulation of these pathways.
PTEN mutations or deletions are strongly associated with various types of cancers including breast and prostate cancer. PTEN interaction with the PI3K/AKT pathway influences cancer development often through loss-of-function mutations leading to unrestrained cellular growth. Beyond cancer PTEN mutations also relate to neurological disorders like Autism Spectrum Disorder where it affects signaling pathways involving proteins like mTOR. Understanding PTEN's role aids in unravelling the mechanistic underpinnings of these diseases paving the way for targeted therapeutic interventions.
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Anti-PTEN antibody [EPR4408-76] ab133532 was shown to react with PTEN in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab255419 (knockout cell lysate Human PTEN knockout HeLa cell lysate ab263829) was used. Wild-type HeLa and PTEN knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. Anti-PTEN antibody [EPR4408-76] ab133532 and Anti-Vinculin antibody [VIN-54] overnight at 4° at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
All lanes: Western blot - Anti-PTEN antibody [EPR4408-76] (Anti-PTEN antibody [EPR4408-76] ab133532) at 1/10000 dilution
Lane 1: Wild-type HeLa cell lysate at 20 µg
Lane 2: PTEN knockout HeLa cell lysate at 20 µg
Lane 2: Western blot - Human PTEN knockout HeLa cell line (ab255419)
Performed under reducing conditions.
Predicted band size: 47 kDa
Observed band size: 47 kDa
Anti-PTEN antibody [EPR4408-76] ab133532 was shown to react with PTEN in wild-type HeLa cells in western blot. The bands observed in PTEN knockout cell line ab255419 (PTEN knockout cell lysate Human PTEN knockout HeLa cell lysate ab263829) below 47 kDa may represent truncated forms and cleaved fragments. This has not been investigated further. HeLa wild-type and PTEN knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with Anti-PTEN antibody [EPR4408-76] ab133532 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.
All lanes: Western blot - Anti-PTEN antibody [EPR4408-76] (Anti-PTEN antibody [EPR4408-76] ab133532) at 1/10000 dilution
Lane 1: Wild-type HeLa cell lysate at 20 µg
Lane 2: PTEN knockout HeLa cell lysate at 20 µg
Lane 2: Western blot - Human PTEN knockout HeLa cell line (ab255419)
Lane 3: HAP1 cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 47 kDa
Observed band size: 47 kDa
Allele-3: Insertion of the selection cassette in exon 5.
Allele-1: 11 bp deletion in exon 5.
Allele-2: 5 bp insertion in exon 5.
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