- 32-5800 - Invitrogen Antibodies PTEN antibody

Submitted references The ISG15-specific protease USP18 regulates stability of PTEN.

Mustachio LM, Kawakami M, Lu Y, Rodriguez-Canales J, Mino B, Behrens C, Wistuba I, Bota-Rabassedas N, Yu J, Lee JJ, Roszik J, Zheng L, Liu X, Freemantle SJ, Dmitrovsky E
Oncotarget 2017 Jan 3;8(1):3-14

Epigenetic and genetic alterations of PTEN in hepatocellular carcinoma.

Wang L, Wang WL, Zhang Y, Guo SP, Zhang J, Li QL
Hepatology research : the official journal of the Japan Society of Hepatology 2007 May;37(5):389-96

In vivo gene therapy of human bladder cancer with PTEN suppresses tumor growth, downregulates phosphorylated Akt, and increases sensitivity to doxorubicin.

Tanaka M, Grossman HB
Gene therapy 2003 Sep;10(19):1636-42

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of PTEN was performed by loading 20 µg of A431 (lane1), HeLa (lane2), NIH/3T3 (lane3) and HEK-293 (lane4) cell lysate using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0301BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (LC5800), and iBlot® Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5 % skim milk for 1 hour at room temperature. PTEN was detected at ~55 kDa using PTEN Mouse monoclonal Antibody (Product # 32-5800) at 1-3 µg/mL in 2.5 % skim milk at 4°C overnight on a rocking platform. Goat Anti-Mouse IgG - HRP Secondary Antibody (Product # 62-6520) at 1:4000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockout of PTEN was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR766883_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of PTEN was performed by loading 30 µg of HeLa Cas9 (Lane 1) andHeLa PTEN KO (Lane 2) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-PTEN Monoclonal Antibody (2F4C9) (Product # 32-5800, 1 µg/mL dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:5,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to PTEN.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of PTEN was done on 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with PTEN Mouse Monoclonal Antibody (Product # 32-5800) at 0.5 µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488 Rabbit Anti-Mouse IgG Secondary Antibody (Product # A-11059) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin (Product # A12381). Panel d is a merged image showing nuclear localization and panel e is a no primary antibody control. The images were captured at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 RPPA-based protein profiling revealed USP18 regulated PTEN levels A. Independent RPPAs are displayed of ED1 and HOP62 lung cancer cell lines stably transfected with a control vector or one of two individual shRNAs against USP18. Signal intensities were normalized and transformed to linear values. RPPA highlighted species showing substantial differences in expression profiles between vector control and USP18 shRNA transfected lung cancer cells are represented by heat-maps for both ED1 and HOP62 lung cancer arrays. Red indicated high and green indicated low protein expression. Cell line clusters are shown as dendrograms. PTEN (red box) was highlighted as one of the proteins showing similar trends in both arrays. B. PTEN expression in the displayed ED1 and HOP62 transfected lung cancer cell lines was quantified using normalized linear values. Cell lines stably transfected with shRNAs against USP18 were compared relative to vector control transfectants. C. Representative immunoblots independently probed with USP18 and PTEN recognizing antibodies confirmed that engineered repression of USP18 decreased PTEN protein levels in ED1 and HOP62 lung cancer cells. PTEN expression was normalized to actin expression and compared between vector control and USP18 shRNA-transfected lung cancer cell lines. D. Decreased PTEN protein levels caused by loss of USP18 expression were rescued by transiently overexpressing GFP-tagged USP18 in ED1 lung cancer cells. Immunoblots were individuall

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Modifying USP18 and ISG15 protein levels altered PTEN protein stability A. Loss of USP18 expression decreased PTEN protein stability. ED1 murine lung cancer cells stably transfected with a vector control or with one of two individual shRNAs against USP18 were subjected to an 8 hour CHX treatment. PTEN expression was quantified relative to respective actin expression at indicated time points and normalized to the 0 hour (before CHX treatment) time point. B. Gain of USP18 expression increased PTEN levels. ED1 and HOP62 lung cancer cells were independently transfected with a USP18 expression plasmid or a control vector. PTEN expression was normalized to actin expression and compared between vector control and USP18 vector-transfected lung cancer cell lines. C. Engineered gain of ISG15 expression decreased PTEN protein levels. ED1 and HOP62 lung cancer cells were independently transfected with ISG15 or a vector control. PTEN was normalized to actin expression and compared between vector control and ISG15 vector-transfected lung cancer cell lines. D. Decreased PTEN levels conferred by overexpressed ISG15 were rescued by transiently overexpressing GFP-tagged USP18 in HOP62 lung cancer cells. Immunoblots were independently probed with GFP and PTEN recognizing antibodies. PTEN protein levels were normalized to actin expression and compared between vector control and ISG15 vector as well as ISG15 vector/USP18 vector-transfected lung cancer cell lines.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 Loss of USP18 expression decreased cytoplasmic PTEN protein A. ED1 and B. HOP62 lung cancer cell lines stably transfected with a vector control or shRNA against USP18 were fixed and stained for PTEN, sodium potassium ATPase, and DAPI. Cells were imaged by confocal microscopy and the magnification was 63X. Percent immunostaining of PTEN in the cytoplasm and nucleus was quantified and compared. Data are representative of 20 cells per group for each analyzed experiment.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 PTEN and USP18 expression profiles are associated in human lung cancers A. Blocking peptide antagonized PTEN immunostaining in the NIH/3T3 (PTEN positive) cell line. B. Representative staining for human lung cancers with low PTEN and USP18 or high PTEN and USP18 expression profiles. All magnifications were 20X. C. PTEN and USP18 immunostaining are positively correlated in human lung cancer ( n = 461) and D. This association was confirmed using a contingency table by grouping negative, low, medium, or high PTEN and USP18 immunostaining profiles in lung cancer cases.

32-5800

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