a549 whole cell lysate Search Results


human a-549 whole cell lysate  (Innovative Research Inc)
N/A
Human A-549 Whole Cell Lysate from Innovative Research is provided as a Liquid. This product is prepared as a positive control for separation (using SDS-PAGE followed by Western Blot analysis). This lysate has been prepared
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human nsclc cell lines a549  (OriGene)
90
OriGene human nsclc cell lines a549
Characterization of <t>A549</t> and A549/DDP cells. (A) A549 and A549/DDP cells were exposed to different concentrations of DDP (1–32 µg/ml) for 24 h, and cell viability was determined by Cell Counting Kit-8 assay. The protein expression levels of (B) E-cadherin, vimentin, α-SMA and (C) CIP2A, p-AKT, AKT, p-mTOR were measured using western blotting; E, epithelial; SMA, smooth muscle actin; p-mTOR, phosphorylated mammalian target of rapamycin; AKT, protein kinase B; DDP, cisplatin; CIP2A, cancerous inhibitor of protein phosphatase 2A; IC 50 , half maximal inhibitory concentration.
Human Nsclc Cell Lines A549, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/a549+whole+cell+lysate/A549+(Human+Lung+Adenocarcinoma)+Whole+Cell+Lysate/pmc06781722-33-0-9
Average 90 stars, based on 1 article reviews
human nsclc cell lines a549 - by Bioz Stars, 2026-10
90/100 stars
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a549 cell lysates  (Rockland Immunochemicals)
90
Rockland Immunochemicals a549 cell lysates
SchB treatment causes a decrease in cell viability after UV irradiation. Relative cell viability was measured by clonogenic assay. <t>A549</t> cells were seeded as 500 cells/60-mm dish, and then incubated for 24 h before the experiment. The cells were subjected to 1 h pre-incubation and 14 days post-incubation ( A ) with SchB (0, 1, 10 and 30 μM) after ( B ) UV irradiation (0, 20 and 50 J/m 2 ) or ( C ) IR irradiation (0, 2 and 6 Gy). Values are expressed as mean ± SD ( n = 3). * P < 0.05 versus SchB untreated control.
A549 Cell Lysates, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/a549+whole+cell+lysate/A549+Whole+Cell+Lysate/pmc02761266-78-6-13
Average 90 stars, based on 1 article reviews
a549 cell lysates - by Bioz Stars, 2026-10
90/100 stars
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a 549 whole cell lysate  (Novus Biologicals)
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human a549 whole cell lysate  (Boster Bio)
N/A
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a549 whole cell lysate  (Aladdin Scientific Corporation)
N/A
The A549 whole cell was prepared using a standard whole cell lysate protocol. The concentration was determined using the BCA assay process.A549 cells are used as an in vitro model for a type II pulmonary
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a549 whole cell lysate  (Aviva Systems)
N/A
Ready-to-use whole cell lysates produced by We are derived from cell lines or tissues using highly refined extraction protocols to ensure exceptionally high quality, protein integrity and lot-to-lot reproducibility. All extracts are tested by SDS-PAGE
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Characterization of A549 and A549/DDP cells. (A) A549 and A549/DDP cells were exposed to different concentrations of DDP (1–32 µg/ml) for 24 h, and cell viability was determined by Cell Counting Kit-8 assay. The protein expression levels of (B) E-cadherin, vimentin, α-SMA and (C) CIP2A, p-AKT, AKT, p-mTOR were measured using western blotting; E, epithelial; SMA, smooth muscle actin; p-mTOR, phosphorylated mammalian target of rapamycin; AKT, protein kinase B; DDP, cisplatin; CIP2A, cancerous inhibitor of protein phosphatase 2A; IC 50 , half maximal inhibitory concentration.

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: Characterization of A549 and A549/DDP cells. (A) A549 and A549/DDP cells were exposed to different concentrations of DDP (1–32 µg/ml) for 24 h, and cell viability was determined by Cell Counting Kit-8 assay. The protein expression levels of (B) E-cadherin, vimentin, α-SMA and (C) CIP2A, p-AKT, AKT, p-mTOR were measured using western blotting; E, epithelial; SMA, smooth muscle actin; p-mTOR, phosphorylated mammalian target of rapamycin; AKT, protein kinase B; DDP, cisplatin; CIP2A, cancerous inhibitor of protein phosphatase 2A; IC 50 , half maximal inhibitory concentration.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Cell Counting, Expressing, Western Blot, Concentration Assay

PPI and PPVII induce cytotoxicity in A549 and A549/DDP cells. (A) Chemical structure of PPI and PPVII. (B) A549 and A549/DDP cells were treated with different dose of PPI (0.5–8 µg/ml) and PPVII (0.5–8 µg/ml) for 24 h, respectively. In A549 cells, the cell inhibition rate of 0.5 µg/ml PPI was significantly different from other concentration groups (**P<0.01); 1.0 µg/ml was significantly different from 4.0 and 8.0 µg/ml (**P<0.01), and 2.0 µg/ml was significantly different from 8.0 µg/ml (**P<0.01). As for A549/DDP cells, the cell inhibition rates of 0.5 µg/ml PPI and 1.0 µg/ml were both significantly different from other concentration groups ( ## P<0.01), and 2.0 µg/ml was significantly different from 8.0 µg/ml ( ## P<0.01). In A549 cells, there were significant differences in cell inhibition rates of PPVII among different concentration groups (**P<0.01); as for A549/DDP cells, there were significant differences among different concentration groups except for 4.0 and 8.0 µg/ml ( ## P<0.01). PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin.

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII induce cytotoxicity in A549 and A549/DDP cells. (A) Chemical structure of PPI and PPVII. (B) A549 and A549/DDP cells were treated with different dose of PPI (0.5–8 µg/ml) and PPVII (0.5–8 µg/ml) for 24 h, respectively. In A549 cells, the cell inhibition rate of 0.5 µg/ml PPI was significantly different from other concentration groups (**P<0.01); 1.0 µg/ml was significantly different from 4.0 and 8.0 µg/ml (**P<0.01), and 2.0 µg/ml was significantly different from 8.0 µg/ml (**P<0.01). As for A549/DDP cells, the cell inhibition rates of 0.5 µg/ml PPI and 1.0 µg/ml were both significantly different from other concentration groups ( ## P<0.01), and 2.0 µg/ml was significantly different from 8.0 µg/ml ( ## P<0.01). In A549 cells, there were significant differences in cell inhibition rates of PPVII among different concentration groups (**P<0.01); as for A549/DDP cells, there were significant differences among different concentration groups except for 4.0 and 8.0 µg/ml ( ## P<0.01). PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Inhibition, Concentration Assay

PPI and PPVII possess chemo-sensitizing effects on A549/DDP cells. (A) A549/DDP cells treated with indicated doses of DDP, or DDP plus either 0.375 µg/ml PPI or 0.75 µg/ml PPI, respectively for 24 h. (B) A549/DDP cells treated with indicated doses of DDP, or DDP plus either 0.375 µg/ml PPVII or 0.75 µg/ml PPVII, respectively for 24 h, cell viability was measured with Cell Counting Kit-8 assay. Data were presented as the mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001 vs. DDP treated group. # P<0.05 and ## P<0.01. PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin.

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII possess chemo-sensitizing effects on A549/DDP cells. (A) A549/DDP cells treated with indicated doses of DDP, or DDP plus either 0.375 µg/ml PPI or 0.75 µg/ml PPI, respectively for 24 h. (B) A549/DDP cells treated with indicated doses of DDP, or DDP plus either 0.375 µg/ml PPVII or 0.75 µg/ml PPVII, respectively for 24 h, cell viability was measured with Cell Counting Kit-8 assay. Data were presented as the mean ± standard deviation. *P<0.05, **P<0.01 and ***P<0.001 vs. DDP treated group. # P<0.05 and ## P<0.01. PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Cell Counting, Standard Deviation

PPI and PPVII reduce the IC 50 value of cisplatin in A549/DDP cells

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII reduce the IC 50 value of cisplatin in A549/DDP cells

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques:

PPI and PPVII enhance DDP-induced apoptosis. A549/DDP cells were treated with DDP (6 µg/ml), PPI (0.75 µg/ml), PPVII (0.75 µg/ml), or DDP (6 µg/ml) in combination with either PPI (0.75 µg/ml) or PPVII (0.75 µg/ml) for 24 h, respectively. Apoptosis of cells was detected by Annexin V-FITC/PI staining and flow cytometry. The apoptotic percentage is shown as a bar graph. Data represents the mean ± standard deviation of three independent experiments. **P<0.01 and ***P<0.001. PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII enhance DDP-induced apoptosis. A549/DDP cells were treated with DDP (6 µg/ml), PPI (0.75 µg/ml), PPVII (0.75 µg/ml), or DDP (6 µg/ml) in combination with either PPI (0.75 µg/ml) or PPVII (0.75 µg/ml) for 24 h, respectively. Apoptosis of cells was detected by Annexin V-FITC/PI staining and flow cytometry. The apoptotic percentage is shown as a bar graph. Data represents the mean ± standard deviation of three independent experiments. **P<0.01 and ***P<0.001. PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Staining, Flow Cytometry, Standard Deviation

PPI and PPVII enhance DDP-induced apoptosis through the P53 pathway and caspases-dependent pathway. A549/DDP cells were treated with DDP (6 µg/ml), PPI (0.75 µg/ml), PPVII (0.75 µg/ml), or DDP (6 µg/ml) in combination with either PPI (0.75 µg/ml) or PPVII (0.75 µg/ml) for 24 h, respectively. The protein expression levels of P53, Bax, Bcl-2 (A), PARP, C-PARP, pro-Caspase-3, C-Caspase-3 (B) were measured using western blotting. PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin; C-PARP, cleaved-poly (ADP-ribose) polymerase 1.

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII enhance DDP-induced apoptosis through the P53 pathway and caspases-dependent pathway. A549/DDP cells were treated with DDP (6 µg/ml), PPI (0.75 µg/ml), PPVII (0.75 µg/ml), or DDP (6 µg/ml) in combination with either PPI (0.75 µg/ml) or PPVII (0.75 µg/ml) for 24 h, respectively. The protein expression levels of P53, Bax, Bcl-2 (A), PARP, C-PARP, pro-Caspase-3, C-Caspase-3 (B) were measured using western blotting. PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin; C-PARP, cleaved-poly (ADP-ribose) polymerase 1.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Expressing, Western Blot

PPI and PPVII reverse EMT and suppress the CIP2A/AKT/mTOR pathway. A549/DDP cells were incubated with DDP (6 µg/ml), PPI (0.75 µg/ml), PPVII (0.75 µg/ml), or DDP (6 µg/ml) in combination with either PPI (0.75 µg/ml) or PPVII (0.75 µg/ml) for 24 h, then cells were harvested for further western blotting analysis. (A) The protein expression levels of E-cadherin, vimentin, α-SMA in A549/DDP cells were measured. (B) The protein expression levels of CIP2A, p-AKT, AKT, p-mTOR, m-TOR were measured. PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin; p-AKT, phosphorylated-protein kinase B; mTOR, mammalian target of rapamycin; CIP2A, cancerous inhibitor of protein phosphatase 2A; SMA, smooth muscle actin; E, epithelial.

Journal: Oncology Letters

Article Title: Polyphyllin I and VII potentiate the chemosensitivity of A549/DDP cells to cisplatin by enhancing apoptosis, reversing EMT and suppressing the CIP2A/AKT/mTOR signaling axis

doi: 10.3892/ol.2019.10895

Figure Lengend Snippet: PPI and PPVII reverse EMT and suppress the CIP2A/AKT/mTOR pathway. A549/DDP cells were incubated with DDP (6 µg/ml), PPI (0.75 µg/ml), PPVII (0.75 µg/ml), or DDP (6 µg/ml) in combination with either PPI (0.75 µg/ml) or PPVII (0.75 µg/ml) for 24 h, then cells were harvested for further western blotting analysis. (A) The protein expression levels of E-cadherin, vimentin, α-SMA in A549/DDP cells were measured. (B) The protein expression levels of CIP2A, p-AKT, AKT, p-mTOR, m-TOR were measured. PPI, Polyphyllin I; PPVII, polyphyllin VII; DDP, cisplatin; p-AKT, phosphorylated-protein kinase B; mTOR, mammalian target of rapamycin; CIP2A, cancerous inhibitor of protein phosphatase 2A; SMA, smooth muscle actin; E, epithelial.

Article Snippet: Human NSCLC cell lines A549 and DDP-resistant A549/DDP cells (OriGene Technologies, Inc.) were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and penicillin (100 U/ml)-streptomycin (100 mg/ml) at 37°C, with 5% CO 2 .

Techniques: Incubation, Western Blot, Expressing

SchB treatment causes a decrease in cell viability after UV irradiation. Relative cell viability was measured by clonogenic assay. A549 cells were seeded as 500 cells/60-mm dish, and then incubated for 24 h before the experiment. The cells were subjected to 1 h pre-incubation and 14 days post-incubation ( A ) with SchB (0, 1, 10 and 30 μM) after ( B ) UV irradiation (0, 20 and 50 J/m 2 ) or ( C ) IR irradiation (0, 2 and 6 Gy). Values are expressed as mean ± SD ( n = 3). * P < 0.05 versus SchB untreated control.

Journal: Nucleic Acids Research

Article Title: Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response

doi: 10.1093/nar/gkp593

Figure Lengend Snippet: SchB treatment causes a decrease in cell viability after UV irradiation. Relative cell viability was measured by clonogenic assay. A549 cells were seeded as 500 cells/60-mm dish, and then incubated for 24 h before the experiment. The cells were subjected to 1 h pre-incubation and 14 days post-incubation ( A ) with SchB (0, 1, 10 and 30 μM) after ( B ) UV irradiation (0, 20 and 50 J/m 2 ) or ( C ) IR irradiation (0, 2 and 6 Gy). Values are expressed as mean ± SD ( n = 3). * P < 0.05 versus SchB untreated control.

Article Snippet: ATM was purified by immunoprecipitation from A549 cell lysates, using anti-ATM monoclonal antibody (Rockland Immunochemicals).

Techniques: Irradiation, Clonogenic Assay, Incubation, Control

SchB treatment abolishes cell cycle checkpoints induced by UV irradiation. ( A ) Effect of SchB on the G2/M checkpoint after UV-induced DNA damage using G2/M synchronized cells. Cell-cycle distributions were analyzed by FACS after 1 h of UV exposure, followed by data analysis using ModFit software. The histogram in gray represents cells synchronized at the G2/M phase by nocodazole. A549 cells were treated with 30 μM SchB or 10 mM caffeine 1 h before UV irradiation (0, 20 and 50 J/m 2 ). The arrow shows the cells distributed in the G1 phase. ( B ) Data were expressed as the percentage of G1 cells in relation to the total number of cells. ( C ) The percentage of mitotic cells was estimated by phosphorylation of histone H3 at Ser10. ( D ) The mitotic percentage of asynchronous cells was estimated by phosphorylation of histone H3 in 10 J/m 2 of UV irradiation followed by 1h incubation. ( E ) The percentage of DNA synthesizing cells was visualized by BrdU intake of cells. ( F ) Data obtained in E were analyzed using XL2 software to determine the percentage of mitotic cells. Values are expressed as mean ± SD ( n = 3). * P < 0.05 versus SchB untreated control.

Journal: Nucleic Acids Research

Article Title: Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response

doi: 10.1093/nar/gkp593

Figure Lengend Snippet: SchB treatment abolishes cell cycle checkpoints induced by UV irradiation. ( A ) Effect of SchB on the G2/M checkpoint after UV-induced DNA damage using G2/M synchronized cells. Cell-cycle distributions were analyzed by FACS after 1 h of UV exposure, followed by data analysis using ModFit software. The histogram in gray represents cells synchronized at the G2/M phase by nocodazole. A549 cells were treated with 30 μM SchB or 10 mM caffeine 1 h before UV irradiation (0, 20 and 50 J/m 2 ). The arrow shows the cells distributed in the G1 phase. ( B ) Data were expressed as the percentage of G1 cells in relation to the total number of cells. ( C ) The percentage of mitotic cells was estimated by phosphorylation of histone H3 at Ser10. ( D ) The mitotic percentage of asynchronous cells was estimated by phosphorylation of histone H3 in 10 J/m 2 of UV irradiation followed by 1h incubation. ( E ) The percentage of DNA synthesizing cells was visualized by BrdU intake of cells. ( F ) Data obtained in E were analyzed using XL2 software to determine the percentage of mitotic cells. Values are expressed as mean ± SD ( n = 3). * P < 0.05 versus SchB untreated control.

Article Snippet: ATM was purified by immunoprecipitation from A549 cell lysates, using anti-ATM monoclonal antibody (Rockland Immunochemicals).

Techniques: Irradiation, Software, Phospho-proteomics, Incubation, Control

Inhibitory effects of SchB treatment on checkpoint proteins. ( A ) A549 cells were pre-incubated with or without 30 μM SchB for 1 h prior to induction of DNA damage. After pre-incubation, cells were subjected to 10 Gy of IR or 20 J/m 2 of UV irradiation, and cultured at 37°C for 1 h and 3 h, respectively. ( B ) Dose-dependent effects of SchB on the phosphorylation level of p53 and Chk1 were observed by adding increasing concentrations of SchB before and after induction of DNA damage by UV irradiation (20 J/m 2 ). A549 cells were incubated with a proteasome inhibitor (50 μM LLnL) throughout the experiments in A, B and C to avoid proteasome-dependent protein degradation. Equal loading of extracted proteins was confirmed by determining immuno-stained tubulin. ( C ) A549 cells were pre-incubated with or without 30 μM SchB or 10 mM caffeine for 1 h prior to induction of DNA damage. After pre-incubation, cells were subjected to UV irradiation (20 J/m 2 ), and cultured at 37°C for 3 h. ( D ) Flag-tagged ATR was expressed in HEK293T cells following transient transfection with a flag-tagged ATR, and the expressed proteins were immunoprecipitated. The association of ATR with ATRIP was analyzed by immunoblot analysis.

Journal: Nucleic Acids Research

Article Title: Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response

doi: 10.1093/nar/gkp593

Figure Lengend Snippet: Inhibitory effects of SchB treatment on checkpoint proteins. ( A ) A549 cells were pre-incubated with or without 30 μM SchB for 1 h prior to induction of DNA damage. After pre-incubation, cells were subjected to 10 Gy of IR or 20 J/m 2 of UV irradiation, and cultured at 37°C for 1 h and 3 h, respectively. ( B ) Dose-dependent effects of SchB on the phosphorylation level of p53 and Chk1 were observed by adding increasing concentrations of SchB before and after induction of DNA damage by UV irradiation (20 J/m 2 ). A549 cells were incubated with a proteasome inhibitor (50 μM LLnL) throughout the experiments in A, B and C to avoid proteasome-dependent protein degradation. Equal loading of extracted proteins was confirmed by determining immuno-stained tubulin. ( C ) A549 cells were pre-incubated with or without 30 μM SchB or 10 mM caffeine for 1 h prior to induction of DNA damage. After pre-incubation, cells were subjected to UV irradiation (20 J/m 2 ), and cultured at 37°C for 3 h. ( D ) Flag-tagged ATR was expressed in HEK293T cells following transient transfection with a flag-tagged ATR, and the expressed proteins were immunoprecipitated. The association of ATR with ATRIP was analyzed by immunoblot analysis.

Article Snippet: ATM was purified by immunoprecipitation from A549 cell lysates, using anti-ATM monoclonal antibody (Rockland Immunochemicals).

Techniques: Incubation, Irradiation, Cell Culture, Phospho-proteomics, Staining, Transfection, Immunoprecipitation, Western Blot

SchB treatment inhibits ATR kinase activity. Kinase activities of ATR ( A ), ATM ( B ), Chk1 ( C ), PI3K ( D ), DNA-PK ( E ) and mTOR ( F ) were measured in vitro as phosphorylation activity in the presence of SchB. A flag-tagged ATR-wt plasmid was transfected into HEK293T cells. ATR protein kinases were purified by immunoprecipitation using anti-Flag M2 antibody and Protein-G agarose. The endogeneous ATM protein was purified from A549 cell lysates with anti-ATM antibody. Kinase activity was monitored for 20 min at 30°C. Chk1 (Upstate), PI3K (Echelon Biosciences), DNA-PK (Promega) and mTOR (Calbiochem) activities were measured using respective assay kits according to the manufacturer ’ s instructions. Values are presented as mean ± SD ( n = 3).

Journal: Nucleic Acids Research

Article Title: Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response

doi: 10.1093/nar/gkp593

Figure Lengend Snippet: SchB treatment inhibits ATR kinase activity. Kinase activities of ATR ( A ), ATM ( B ), Chk1 ( C ), PI3K ( D ), DNA-PK ( E ) and mTOR ( F ) were measured in vitro as phosphorylation activity in the presence of SchB. A flag-tagged ATR-wt plasmid was transfected into HEK293T cells. ATR protein kinases were purified by immunoprecipitation using anti-Flag M2 antibody and Protein-G agarose. The endogeneous ATM protein was purified from A549 cell lysates with anti-ATM antibody. Kinase activity was monitored for 20 min at 30°C. Chk1 (Upstate), PI3K (Echelon Biosciences), DNA-PK (Promega) and mTOR (Calbiochem) activities were measured using respective assay kits according to the manufacturer ’ s instructions. Values are presented as mean ± SD ( n = 3).

Article Snippet: ATM was purified by immunoprecipitation from A549 cell lysates, using anti-ATM monoclonal antibody (Rockland Immunochemicals).

Techniques: Activity Assay, In Vitro, Phospho-proteomics, Plasmid Preparation, Transfection, Purification, Immunoprecipitation

SchB prevents ATR-dependent signaling pathway after DNA damage. ( A ) AT2KY fibroblasts were pre-incubated with or without 30 μM SchB for 1 h, and then subjected to UV irradiation (20 J/m 2 ). The cells were harvested 4 h later and phosphorylation levels of p53, Chk1, SMC1 and BRCA1 were measured to assess checkpoint function. ( B ) A549 cells were transfected with siRNA for control (siGFP), ATM or ATR using the oligofectamine method, and the cell extracts were immunoblotted with anti-phosphorylated p53, phosphorylated Chk1, total-ATM, total-ATR or anti-tubulin. After 72 h incubation for the knockdown of ATM or ATR, cells were subjected to UV irradiation (20 J/m 2 ) followed by 4 h incubation with or without SchB (30 μM) pre/post-incubation. The equal loading of an extracted protein was confirmed using anti-monoclonal tubulin antibody. ( C ) The cell viability was measured in ATR-deficient Seckel syndrome fibroblasts with or without SchB (0, 1, 10 and 30 μM) after UV irradiation (0, 25, 50 and 75 J/m 2 ). Values are expressed as mean ± SD ( n = 3).

Journal: Nucleic Acids Research

Article Title: Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response

doi: 10.1093/nar/gkp593

Figure Lengend Snippet: SchB prevents ATR-dependent signaling pathway after DNA damage. ( A ) AT2KY fibroblasts were pre-incubated with or without 30 μM SchB for 1 h, and then subjected to UV irradiation (20 J/m 2 ). The cells were harvested 4 h later and phosphorylation levels of p53, Chk1, SMC1 and BRCA1 were measured to assess checkpoint function. ( B ) A549 cells were transfected with siRNA for control (siGFP), ATM or ATR using the oligofectamine method, and the cell extracts were immunoblotted with anti-phosphorylated p53, phosphorylated Chk1, total-ATM, total-ATR or anti-tubulin. After 72 h incubation for the knockdown of ATM or ATR, cells were subjected to UV irradiation (20 J/m 2 ) followed by 4 h incubation with or without SchB (30 μM) pre/post-incubation. The equal loading of an extracted protein was confirmed using anti-monoclonal tubulin antibody. ( C ) The cell viability was measured in ATR-deficient Seckel syndrome fibroblasts with or without SchB (0, 1, 10 and 30 μM) after UV irradiation (0, 25, 50 and 75 J/m 2 ). Values are expressed as mean ± SD ( n = 3).

Article Snippet: ATM was purified by immunoprecipitation from A549 cell lysates, using anti-ATM monoclonal antibody (Rockland Immunochemicals).

Techniques: Incubation, Irradiation, Phospho-proteomics, Transfection, Control, Knockdown

Phosphorylation of ERK at Thr202 and Tyr204. A549 cells were pre-treated with 50 μM PD098059 or 30 μM SchB after serum starvation for 16 h. The phosphorylation of ERK was activated by adding 100 ng/ml PMA followed by a 5 min incubation. The cells were lysed and applied to immunoblot analysis. Equal loading of an extracted protein was confirmed using anti-monoclonal tubulin antibody.

Journal: Nucleic Acids Research

Article Title: Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response

doi: 10.1093/nar/gkp593

Figure Lengend Snippet: Phosphorylation of ERK at Thr202 and Tyr204. A549 cells were pre-treated with 50 μM PD098059 or 30 μM SchB after serum starvation for 16 h. The phosphorylation of ERK was activated by adding 100 ng/ml PMA followed by a 5 min incubation. The cells were lysed and applied to immunoblot analysis. Equal loading of an extracted protein was confirmed using anti-monoclonal tubulin antibody.

Article Snippet: ATM was purified by immunoprecipitation from A549 cell lysates, using anti-ATM monoclonal antibody (Rockland Immunochemicals).

Techniques: Phospho-proteomics, Incubation, Western Blot