34-1500

antibody from Invitrogen Antibodies

Targeting: PTTG1 EAP1, HPTTG, PTTG, securin, TUTR1

Provider product page for 34-1500

Western blot

ELISA

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [22]
Comments [0]
Validations
Immunocytochemistry [1]
Other assay [31]

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Product number: 34-1500 - Provider product page
Provider: Invitrogen Antibodies
Product name: Securin Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Reactivity: Human
Host: Rabbit
Isotype: IgG
Vial size: 100 µg
Concentration: 0.25 mg/mL
Storage: -20°C

PTTG1 protein structure - 34-1500 shown in red.

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Experimental details: Fig. 2. MASTL-mediated PP2A inhibition in mitosis is essential to direct cyclin B1 destruction by the APC/C. (A) Forced mitotic exit by either Aurora B or Cdk1 inhibition stabilises cyclin B1 in MASTL RNAi cells. HeLa cells were synchronised in mitosis by thymidine synchronisation and taxol treatment. Mitotic cells were treated with 10 uM of RO-3306 (Cdk1 inhibitor) or 1 uM of AZD1152 (Aurora B inhibitor) for 90 minutes to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. (B) APC/C phosphorylation in mitosis critically depends on cyclin B1-Cdk1 activity. HeLa cells transfected with indicated siRNA pools were synchronised in mitosis by a thymidine block followed by subsequent taxol treatment. Cells arrested in mitosis were collected by mitotic shake-off. Extracts were processed for western blot analysis either 24 hours ( PLK1 RNAi) or 48 hours after the siRNA transfection and blotted for the proteins as indicated. (C) Enforcing mitotic exit in absence of MASTL severely impairs the proteolysis of cyclin B1. HeLa cells transfected with indicated siRNAs were synchronised in mitosis by thymidine synchronisation and taxol treatment. Mitotic cells were treated with 10 uM of RO-3306 for indicated time points to obtain cells in G1 phase. Extracts were processed for western blot analysis and blotted for indicated proteins. The intensity of the cyclin B1, geminin, and securin signals were quantified for each condition and cor

Supportive validation

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Experimental details: Fig. 3. MASTL depletion significantly impairs cyclin B1 recruitment to the mitotic APC/C. (A) Cyclin B1 binds to the APC/C specifically in mitosis. HeLa cells were synchronised in G2 and M phase by a thymidine block, followed by nocodazole or taxol treatment. Cells arrested in mitosis were collected by mitotic shake-off, and treated for another 2 hours with 5 uM MG132 where indicated. Cell extracts were subjected to immunoprecipitation using a goat anti-APC4. Aliquots of the immunoprecipitates were analysed by western blotting. (B) MASTL silencing impairs the recruitment of cyclin A2 and cyclin B1 to the mitotic APC/C. HeLa cells transfected with indicated siRNAs were synchronised in G2 phase and mitosis using thymidine and taxol. Cells arrested in mitosis were obtained by mitotic shake-off and lysed directly or subsequently treated with 10 uM of RO-3306 for 90 minutes to obtain G1 phase cells. Cell extracts were subjected to immunoprecipitation using a goat anti-APC4 and aliquots of the immunoprecipitates were analysed by western blotting. Note that equal amounts of APC4 are immunoprecipitated in the mitotic fractions, whereas cyclins A2 and B1 bound to the APC/C are hardly detectable in the MASTL RNAi extracts. The asterisk (*) indicates the remaining securin signal after reprobing the blot with anti-geminin (right-hand panel). (C) Direct binding of cyclin B1 to the APC/C may depend on extensive APC3 phosphorylation in mitosis. HeLa cells treated as in (B), were subjected t

Supportive validation

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Experimental details: Fig. 4. Cyclin B1 associates with the mitotic APC/C, independent of Cdc20. (A) Cyclin B1 recruitment to the APC/C does not involve direct Cdc20 binding, different from cyclin A2. HeLa cells transfected with indicated siRNAs were synchronised in G2 phase and mitosis using thymidine and taxol. Cells arrested in mitosis were obtained by mitotic shake-off and lysed directly or subsequently treated with 10 uM of RO-3306 for 90 minutes to obtain G1 phase cells. Cell extracts were subjected to immunoprecipitation using a rabbit anti-Cdc20 and aliquots of the immunoprecipitates were analysed by western blotting. Asynch refers to an asynchronous, non-transfected lysate. (B) Genetic ablation of CDC20 results in a mitotic arrest due to cyclin B1 stabilisation. Cdc20(Delta/Delta); RERT(+/Cre) mouse embryonic fibroblasts (MEFs) were asynchronously grown or thymidine synchronised for 9 hours and treated with 1 uM of 4-hydroxytamoxifen (4-OHT) to induce Cre activity, resulting in the excision of exon 2 of CDC20 (Cdc20(Delta) allele). Cells arrested in mitosis by the 4-OHT treatment were obtained by mitotic shake-off. Extracts of asynchronous or 4-OHT treated cells were subjected to western blot analysis. The dashed line indicates where two lanes have been placed next to each other. (C) Cyclin B1 binds the mitotic APC/C regardless of the presence of Cdc20. Cdc20(Delta/Delta); RERT(+/Cre) MEFs were either asynchronously grown or synchronised in mitosis using thymidine and nocodazole or 4-OHT

Supportive validation

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Experimental details: Fig. 5. MASTL depletion increases the frequency of anaphase bridges that may depend on the activity of separase. (A) DNA bridges formed during anaphase are specific to MASTL silencing. Asynchronously growing HeLa cells were transfected with indicated siRNAs, formaldehyde fixed after 48 hours, and subjected to immunofluorescence staining using anti-alpha-Tubulin and DAPI. Arrows indicate DNA bridges connecting the daughter cells in G1 phase. (B) Combined depletion of MASTL and PPP2CA prevents the formation of DNA bridges. HeLa cells stably infected with an shRNA targeting PPP2CA (sh PPP2CA ) or a control shRNA (sh GFP ) were transfected with indicated siRNAs. Cells were formaldehyde fixed after 48 hours, and subjected to immunofluorescence staining using DAPI. Representative images show a regular cell or two daughter cells that are connected to each other by a DNA bridge (indicated by the red arrow). The amount of cells containing a DNA bridge were scored and quantified for each condition [ n = 3; mean+-standard deviation (s.d.)]. (C) Western blot analysis to show the effects of indicated shRNAs on total protein levels. A reduction in the protein levels of PP2A-A and PP2A-Calpha demonstrates the knockdown efficiency of sh PPP2CA . (D) MASTL silencing does not alter the mitotic chromosome morphology. HeLa cells transfected with indicated siRNAs were synchronised in mitosis by a thymidine block followed by subsequent nocodazole treatment for 5 hours. Cells were processed for chr

Supportive validation

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Experimental details: Figure 1 hPTTG1 overexpression reinforces senescence and senescence-associated secretory phenotype (SASP) . (A) Cell lysates of MDA-MB-231, MCF-7, and MCF-10A cells were collected for immunoblot analysis. (B) MCF-7 cells stably transfected with pcDNA3.1 (Mock; MCF-7 Mock ) or pcDNA3.1- hPTTG1 (hPTTG1; MCF-7 hPTTG1 ) were orthotopically injected into fourth mammary fat pads of SCID mice. The primary tumor weights were measured 18 weeks after implantations. Representative tumors are shown in the upper panel. Quantitative results are shown in the lower panel ( n = 6 per group). The results are presented as the mean +- SEM. * P < 0.05. (C) Tumor growth was measured weekly after orthotopic implantations of MCF-7 cells stably transfected with pcDNA3.1 (Mock) or pcDNA3.1- hPTTG1 (hPTTG1). The experiments were terminated 18 weeks after implantation. Representative tumors are shown in (B). n = 6 per group per time point. Each time point shows the mean +- SEM. * P < 0.05. (D) MCF-7 and MCF-10A cells stably transfected with pcDNA3.1 (Mock) or pcDNA3.1- hPTTG1 (hPTTG1) were plated for the senescence assay. The percentage of positively stained cells is indicated to the right of the respective images. Data represent mean +- SEM ( n = 3). *** P < 0.0001. (E) Cell lysates were collected from MCF-7 and MCF-10A cells stably transfected with pcDNA3.1 (Mock) or pcDNA3.1- hPTTG1 (hPTTG1) for immunoblot analysis. WT, untransfected cells. (F) Conditioned media were collected from MCF-7 cells stably

Supportive validation

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Experimental details: Figure 7 Positive correlation between hPTTG1, CXCR2, and p21 expression in human breast invasive ductal carcinomas (IDCs) . (A) The expression pattern of hPTTG1, CXCR2, and p21 in representative tumor tissues of patients with breast IDC were immunohistochemically analyzed. Tissues were scored as having strong (3 + ), moderate (2 + ) or weak expression (1 + /0 + ). The criteria are described in the Materials and methods. Original magnification, x400. Scale bars, 25 mum. (B-D) The expression data for hPTTG1, CXCR2, and p21 in 100 IDC specimens. The correlation between the indicated proteins was analyzed by using the Pearson chi 2 test. (E) The expression data for p21 in 84 IDC specimens in which hPTTG1 expression was relevant to CXCR2 expression (the 84 specimens are marked with boxes in (B). (F) The expression data of hPTTG1 and CXCR2 in 50 primary IDC specimens and 50 matched metastatic carcinomas (upper panel). The specimens were classified into four groups: hPTTG1(2 + ,3 + )/CXCR2(2 + ,3 + ), hPTTG1(2 + ,3 + )/CXCR2(1 + ,0 + ), hPTTG1(1 + ,0 + )/CXCR2(2 + ,3 + ), and hPTTG1(1 + ,0 + )/CXCR2(1 + ,0 + ). The percentage of hPTTG1/CXCR2-stained specimens of each group is indicated in the lower panel. (G) The expression pattern of hPTTG1 and CXCR2 in representative breast IDC and matched metastatic lymph nodes (LNs). Original magnification, x400. Scale bars, 25 mum. (H) MDA-MB-231 cells were stained with FITC-conjugated anti-CXCR2 or isotype control IgG for FACS analysis. (I) Ce

Supportive validation

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Experimental details: PTTG1 is upregulated with KLF6 knockdown. HepG2 cells were transfected with siKLF6 or siControl and RNA and protein collected after 24 h. (A) qRT-PCR on cDNA showed 57% decrease in KLF6 mRNA, with a corresponding 3.7-fold increase in PTTG1. (B) Protein extracts were submitted to Western blot analysis and approximately a 50% decrease in KLF6 protein levels resulted in approximately twofold overexpression of PTTG1. The data presented in (B) is representative of at least three independent experiments.