33-1700

antibody from Invitrogen Antibodies

Targeting: PLK1 PLK

Provider product page for 33-1700

Western blot

ELISA

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [41]
Comments [0]
Validations
Immunocytochemistry [1]
Flow cytometry [1]
Other assay [32]

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Product number: 33-1700 - Provider product page
Provider: Invitrogen Antibodies
Product name: PLK1 Antibody Cocktail
Antibody type: Monoclonal
Antigen: Other
Description: 33-1700 contains a cocktail of monoclonal mouse PLK1 containing clone PL6, isotype IgG1, kappa and clone PY20, isotype IgG2b, kappa.
Reactivity: Human, Mouse, Rat, Xenopus
Host: Mouse
Isotype: IgG
Antibody clone number: PL6/PL2
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: -20°C

PLK1 protein structure - 33-1700 shown in red.

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Experimental details: Figure 5. Pharmacological inhibition of PLK1 alters protein levels of autophagy andBcl - 2 family member proteins. 621-101 cells were treated for24 hours with the indicated concentrations of BI-2536, and the lysates wereimmunoblotted with the indicated antibodies.

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Experimental details: Figure 2 Cell cycle regulatory factors following exposure to ionizing radiation (IR) . Four colorectal carcinoma cell lines (HCT116, Co115, SW620, and KM20L2) were exposed (+) to 8 Gy of IR, or left unexposed (-), and further incubated for the indicated time periods before analysis. Upper panel: Protein expression levels of Plk1, p53, Cyclin D1, and p21 were analyzed by Western blot immunostaining, using alpha-tubulin as protein loading control. Lower panel: mRNA expression levels of PLK , CCND1 , and CDKN1A were analyzed by Northern blot hybridization, using 18S rRNA as RNA loading control.

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Experimental details: Figure 5 Cell cycle regulatory factors upon TSA treatment . The HCT116 and SW620 cell lines were treated (+) with 100 nM TSA, or left untreated (-), and further incubated for the indicated time periods before analysis. Upper panel: Protein expression levels of Plk1, p53, Cyclin D1, and p21 were analyzed by Western blot immunostaining, using alpha-tubulin as protein loading control. Lower panel: mRNA expression levels of PLK , CCND1 , and CDKN1A were analyzed by Northern blot hybridization, using 18S rRNA as RNA loading control.

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Experimental details: Figure 3 MDCs express cyclin B1 and the active form of Cdk1 ( a ) Cells were grown on coverslips and either not treated (upper row) or treated with CPT for 48 h (lower row), stained with DAPI (blue) and with anti-(cyclin B1) antibodies (red). Cells were analysed by immunofluorescence microscopy. Scale bar, 50 mum. ( b ) Extracts were prepared from cells either not treated (NT) or treated with CPT. CPT-treated cells were analysed further as total cultures (TDC) or cultures fractionated by mechanical shake-off into adherent interphase cells (IDC) or rounded cells (MDC). Extracts were also prepared from cells treated with nocodazole (Noco). Samples were processed by Western blotting with antibodies against cyclin B1, Cdk1, phospho-Tyr 15 Cdk1 (P-Y15 Cdk1), phospho-Thr 320 PP1a (P-T320 PP1), Plk1, phospho-Thr 210 Plk1 (P-T210 Plk1) or actin. Molecular masses are indicated in kDa.

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Experimental details: FIGURE 7: (A) Phosphorylation of Wee1 at Ser-139 is required for normal Wee1 turnover. HEK293T cells were transfected with HA-tagged, wild-type Wee1 (wt) or the different HA-tagged Wee1 mutants (S123A, S139A, S139D, and S123/139A or 2A). At 24 h posttransfection, cells were treated with cycloheximide to inhibit protein synthesis and then harvested at the indicated time points to check HA-Wee1 protein levels by immunoblotting. HA-Wee1 bands were quantified and normalized with respect to actin. HA-Wee1 protein levels from 0 h were considered as 1. The data are representative of three independent experiments. (B) Cdc14A dephosphorylates Wee1 at Ser-123 and Ser-139 to regulate Wee1 stability. U-2-OS cells stably expressing retrovirally transduced, HA-tagged Wee1 wt or the double 2A mutant were treated with control or Cdc14A siRNAs for 48 or 72 h. Cellular extracts were obtained and analyzed by immunoblotting against the indicated proteins. These blots are representative of three independent experiments. (C) Recombinant HA-Wee1 proteins (wt, S123A, S139A, or 2A mutants) were expressed in HEK293T cells, immunoprecipitated with anti-HA antibodies, and analyzed by immunoblotting with the indicated antibodies. This experiment was performed twice.

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Experimental details: Figure 1 NFBD1 and PLK1 are both expressed in the G2/M phase and form a protein complex. (A) HeLa cells were double-thymidine blocked at the late G1 phase and then released into fresh medium. At the indicated times after release, cells were stained with propidium iodide (PI) and analyzed by FACS. (B) Schematic representation of cell-cycle distributions under the abovementioned experimental conditions. (C) Western blot analysis. At the indicated time after release, whole cell lysates were prepared and immunoblotted against anti-NFBD1 (first panel), anti-PLK1 (second panel), or anti-actin (third panel) antibody. Immunoblotting for actin is shown as a control for protein loading.

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Experimental details: Figure 2 Co-immunoprecipitation and co-localization between NFBD1 and PLK1 in the G2/M phase. (A) Immunoprecipitation analysis. HeLa cells were synchronized by the double-thymidine block regimen. Six hours after the second release, whole cell lysates prepared from HeLa cells were immunoprecipitated with normal rabbit serum or with a polyclonal anti-NFBD1 antibody followed by immunoblotting with a monoclonal anti-PLK1 antibody (left panel). Reciprocal experiments using normal mouse serum and a monoclonal anti-PLK1 antibody are shown in the middle panel. (B) Indirect immunofluorescence staining. HeLa cells were synchronized by the double-thymidine block regimen. Six hours after the second release, mitotic cells were simultaneously stained with polyclonal anti-NFBD1 (green) and monoclonal anti-PLK1 antibodies (red). Merged images (yellow) indicate the co-localization of NFBD1 with PLK1. Nuclear DNA was stained with DAPI (blue).

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Experimental details: Figure 6 Knockdown of NFBD1 accelerates G2/M progression. (A) siRNA-mediated knockdown of NFBD1 results in a reduction of endogenous NFBD1. HeLa cells were synchronized by the double-thymidine block regimen and transfected with 10 nM of NFBD1 siRNA or control siRNA at the first release. At the time of the second release, whole cell lysates prepared from transfected cells were subjected to immunoblotting with anti-NFBD1 antibody (top panel). Western blotting for actin is shown as a control for protein loading (bottom panel). (B) Western blotting analysis of mitosis-related proteins. Whole cell lysates were prepared in NFBD1 siRNA transfected cells (right panel) or control siRNA transfected cells (left panel) at the indicated times after the second release and immunoblotted with anti-phospho-histone H3 antibody, anti-PLK1 antibody, anti-cyclin A antibody, anti-cyclin B antibody, or anti-actin antibody. (C) Cumulative percentages of NEBD-positive cells. GFP-cyclin B-expressing HeLa cells were synchronized and transfected with the indicated siRNAs. The times of NEBD were defined by the loss of a defined nuclear boundary and nuclear accumulation of cyclin B in the NFBD1 siRNA-transfected cells (n= 45) and control siRNA-transfected cells (n= 56). The significant difference was demonstrated by the Student's t -test.

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Experimental details: Figure 1. PLK1 protein levels are increased in hamartin and tuberin deficient cells and arerapamycin-sensitive. ( A ) Lysates from exponentially growing Tsc1 +/- , Tsc1 -/- , Tsc2 +/+ / Tp53 -/- and Tsc2 -/- / Tp53 -/- MEF were immunoblotted with the indicated antibodies. ( B ) Lysates from Tsc1 -/- MEF transduced with vector(208-P2) or TSC1 (208-T3), and from tuberin-deficient ELT3 cells transduced withvector (ELT3-V3) or TSC2 (ELT3-T3) were immunoblotted with the indicated antibodies.( C ) Lysates from Tsc1 +/- and Tsc1 -/- MEF treated for 24 hours with0, 50 or 100 nM rapamycin were immunoblotted with the indicated antibodies.PLK1 protein levels normalized to beta-actin and to vehicle-treated Tsc1 +/- MEF are indicated below the PLK1immunoblots.

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Experimental details: Figure 5. TOPBP1 interacts with PLK1. Immunoblots of control, anti-PLK1 (A) or anti-TOPBP1 (B) immunoprecipitates (IPs) from U2OS cells treated or not with nocodazole (40 ng/ml) for 4 h before collection. (C) Immunoblots of control and anti-PLK1 IPs from U2OS cells treated or not with CalAM (4 uM) or nocodazole (40 ng/ml) for 4 h before collection. (D) Model of TOPBP1 role in HR.

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Experimental details: Figure 4. Pharmacological inhibition of PLK1 induces apoptosis in hamartin and tuberindeficient cells and attenuates autophagy. ( A ) Phase contrastmicrographs of hamartin deficient 208-P2 MEF and hamartin re-expressing 208-T3 MEFtreated with vehicle control (DMSO) or 30 nM BI-2536 for 3 days. Scale bar400 mum. ( B ) Tuberin deficient ELT3-V3 and tuberin re-expressingELT3-T3 cells were treated with vehicle control (DMSO), 30 nM BI-2536, or20 nM rapamycin for 1, 2 and 3 days, and lysates were immunoblotted withthe indicated antibodies. Asterisk (*) on tuberin immunoblot indicates anon-specific band identified on rat species with the anti-tuberin antibody used, andwas previously described. 50 ( C ) WI38 primary fibroblasts ectopically expressing GFP-LC3 weretreated with vehicle control (DMSO), 20 nM rapamycin, or 100 nM BI-2536for 24 hours, fixed, and the number of GFP-LC3 punctae per nucleus wasquantified from epi-fluorescence digital micrographs. n >= 9,** and *** indicate P < 0.01 and P < 0.001, respectively, compared to DMSO. ( D )HEK293 cells were pre-treated for 24 hours with 0.1% v/v DMSO, or100 nM BI-2536, or 70 ng/ml nocodazole, media were removed, cellmonolayers were washed twice with HBSS, incubated for the indicated time with HBSSin the presence of pre-treatment compounds, lysed, and lysates were immunoblottedfor p62 and beta-actin. The p62 / beta-actin ratios (normalized for the 0 htime-point within each treatment group) are shown below the p62 immunoblots and atth

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5. Pharmacological inhibition of PLK1 alters protein levels of autophagy andBcl - 2 family member proteins. 621-101 cells were treated for24 hours with the indicated concentrations of BI-2536, and the lysates wereimmunoblotted with the indicated antibodies.

Supportive validation

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Experimental details: Figure 4 Formaldehyde Selectively Depletes Components of the Cellular Proteome (A) Abundance of proteins involved in homologous recombination or the Fanconi anemia repair pathway in wild-type HeLa Kyoto cells treated with FA for 5 hr. (B) Abundance of proteins involved in non-homologous end-joining in HeLa Kyoto cells treated with FA for 5 hr. (C) Volcano plot showing the results of the SWATH-MS analysis of HeLa Kyoto cells treated with or without 200 muM FA for 5 hr. Each dot represents a protein with Benjamini-Hochberg adjusted p values plotted along the y axis, and the fold change in abundance following FA treatment along the x axis. The horizontal black line indicates where p = 0.05. Red dots mark proteins that are depleted by >=25% compared to untreated controls in a statistically significant manner (p < 0.05). Proteins tested by western blotting are labeled. (D) Abundance of selected proteins from SWATH-MS analysis in HeLa Kyoto cells treated with FA for 5 hr. See also Figure S4 and Tables S1 and S2 .

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Experimental details: Figure 4 Differential engagement of PLK1 PBD substrates via the Tyr pocket. ( A ) FRAP analysis of interphase centrosomes in live cells. GFP-PLK1 Wt/AAD/AM at the centrosome was photobleached and its recovery monitored at 0.2 s intervals. The median t 1/2 time taken for 50% maximal recovery for GFP-PLK1 Wt/AAD/AM were found to be 0.7 s, 0.4 s and 0.3 s respectively. The arrowhead indicates the point of photobleaching. An inset shows data points for the first 5 seconds including photobleaching and recovery time of GFP signal. ( B ) HeLa cells expressing GFP-PLK1 Wt/AAD/AM were synchronized in mitosis by double thymidine block and released as shown in the experimental schedule. The cell lysates were immunoprecipitated using GFP-Trap(r) beads to pull down GFP-PLK1 Wt/AAD/AM and analysed by immunoblotting. The data presented is representative of two independent experiments. ( C ) HeLa cells expressing GFP-PLK1 Wt/AAD/AM were transfected with PBIP1 Wt -V5 or pcDNA(tm)3.1/V5-HisA vector (or V5 vector) and harvested 24 h later. PBIP1 Wt -V5/ V5 was pulled down from the lysates using V5 antibody and immunoprecipitates were analysed by immunoblotting. Asterisks (*) indicate cross-reacting bands. The data presented is representative of two independent experiments. ( D ) HeLa GFP-PLK1 Wt overexpressing cells were transfected with either PBIP1 Wt/F71A/T78A -V5 or V5 vector and harvested 24 h later. PBIP1 Wt/F71A/T78A -V5 was pulled down from the lysates using V5 antibody and analysed by