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    TaKaRa hela tet off cells
    The SGT1-HSP90 complex contributes to CENP-A monoubiquitylation and diubiquitylation in vivo. (A) Representative images of the in vivo ubiquitylation assay with the combination of SGT1 siRNA (#1 + #2), CUL4A siRNA (#1), or Luc siRNA control (Table S2; see CENP-A in vivo ubiquitylation assay in Materials and Methods). <t>HeLa</t> <t>Tet-Off</t> cells were cotransfected with the indicated constructs and siRNAs. Proteins in 5% of the total cell lysates (Input) and immunoprecipitates (IP) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (**) Putative di-Ub-CENP-A-Flag, (*) Putative mono-Ub-CENP-A-Flag. (B) Representative images of the in vivo ubiquitylation assay performed as (A) with the combination of HSP90 siRNA (#1) or Luc siRNA control (Table S2; see CENP-A in vivo ubiquitylation assay in Materials and Methods).
    Hela Tet Off Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    TaKaRa hela tet on cells
    CDT2 regulates CHK1 turnover in response to replication stress. (A and B) HU-induced CHK1 turnover in CDT2-depleted cells. <t>HeLa</t> cells transfected with control siRNA (GL3) or siRNA specific for CDT2 were cultured for 48 h and then incubated in medium containing 5 mM HU in the absence or presence of 10 μM MG132. At the indicated times post-HU treatment, cells were harvested and analyzed by Western blotting. A representative experiment is shown in panel A, and the quantitation of data from 5 independent experiments is shown in panel B. (C and D) UV-induced CHK1 turnover in CDT2-depleted cells. HeLa cells transfected with control siRNA or siRNA specific for CDT2 were exposed to 50 J/m 2 UV at 48 h posttransfection. Cells were then cultured in the absence or presence of 10 μM MG132. At the indicated times post-UV exposure, cells were harvested and analyzed by Western blotting. A representative experiment is shown in panel C, and the quantitation of data from 4 independent experiments is shown in panel D. (E) Prolonged half-life of CHK1 in CDT2-depleted cells. HeLa <t>Tet-on</t> cells were transfected with a plasmid encoding Tet-inducible Flag-CHK1 followed by either control siRNA or siRNA specific for CDT2. Cells were cultured in the presence of 0.2 μg/ml doxycycline for 14 h to induce Flag-CHK1 expression. Cells were washed, cultured for 4 h in fresh medium, and then cultured in medium containing 5 mM HU. At the indicated times post-HU treatment, cells were harvested and analyzed by Western blotting. Levels of Flag-CHK1 are shown relative to levels at the 0-h time points (lanes 1 and 4). (F) Effects of CDT1 overexpression on CHK1 stability. HeLa cells expressing Flag-tagged CDT1 for 24 h were incubated in the absence or presence of 5 mM HU for 6 h. Cell lysates were analyzed by Western blotting. The arrowhead indicates endogenous CDT1.
    Hela Tet On Cells, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Image Search Results


    The SGT1-HSP90 complex contributes to CENP-A monoubiquitylation and diubiquitylation in vivo. (A) Representative images of the in vivo ubiquitylation assay with the combination of SGT1 siRNA (#1 + #2), CUL4A siRNA (#1), or Luc siRNA control (Table S2; see CENP-A in vivo ubiquitylation assay in Materials and Methods). HeLa Tet-Off cells were cotransfected with the indicated constructs and siRNAs. Proteins in 5% of the total cell lysates (Input) and immunoprecipitates (IP) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (**) Putative di-Ub-CENP-A-Flag, (*) Putative mono-Ub-CENP-A-Flag. (B) Representative images of the in vivo ubiquitylation assay performed as (A) with the combination of HSP90 siRNA (#1) or Luc siRNA control (Table S2; see CENP-A in vivo ubiquitylation assay in Materials and Methods).

    Journal: Cell Cycle

    Article Title: SGT1-HSP90 complex is required for CENP-A deposition at centromeres

    doi: 10.1080/15384101.2017.1325039

    Figure Lengend Snippet: The SGT1-HSP90 complex contributes to CENP-A monoubiquitylation and diubiquitylation in vivo. (A) Representative images of the in vivo ubiquitylation assay with the combination of SGT1 siRNA (#1 + #2), CUL4A siRNA (#1), or Luc siRNA control (Table S2; see CENP-A in vivo ubiquitylation assay in Materials and Methods). HeLa Tet-Off cells were cotransfected with the indicated constructs and siRNAs. Proteins in 5% of the total cell lysates (Input) and immunoprecipitates (IP) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (**) Putative di-Ub-CENP-A-Flag, (*) Putative mono-Ub-CENP-A-Flag. (B) Representative images of the in vivo ubiquitylation assay performed as (A) with the combination of HSP90 siRNA (#1) or Luc siRNA control (Table S2; see CENP-A in vivo ubiquitylation assay in Materials and Methods).

    Article Snippet: To study the interaction of exogenous CUL4A-Flag with endogenous COPS8 , HeLa Tet-Off cells were cotransfected with plasmids pTRM4-CUL4A-Flag and pCGN-HA-Ubiquitin (Table S3; see Cell culture and transfection).

    Techniques: In Vivo, Ubiquitin Assay, Construct, Western Blot

    SGT1-HSP90 complex is required for composition of the CUL4A complex and recognition of CENP-A by COPS8. (A) SGT1A interacts with CUL4A in vivo. Immunoblot analysis of CUL4A immunoprecipitates. Plasmids pTRM4-SGT1A-Flag and pcDNA3-myc3-CUL4A (Table S3) were cotransfected into HeLa Tet-Off cells. Forty-eight hours after transfection, protein extracts were prepared. Proteins in 1% of the total cell lysate (Input) and precipitate (IP) obtained by using anti-c-myc antibody (Table S1) were detected by Western blot analysis using anti-Flag antibody. (B) Interaction between CENP-A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of endogenous CENP-A immunoprecipitates. Indicated siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa cells. Seventy-two hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using rabbit polyclonal anti-CENP-A antibody (Table S1) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (C) Interaction between CUL4A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of CUL4A immunoprecipitates. Plasmid pTRM4-CUL4A-Flag (Table S3) and indicated siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa Tet-Off cells. Seventy-two hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using ANTI-FLAG M2 Affinity Gel (SIGMAALDRICH) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (D) Interaction between CUL4A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of COPS8 immunoprecipitates. The indicated plasmids (Table S3) and siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa Tet-Off cells. Forty-eight hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using Anti-HA.11 Epitope Tag Affinity Matrix (Covance) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input).

    Journal: Cell Cycle

    Article Title: SGT1-HSP90 complex is required for CENP-A deposition at centromeres

    doi: 10.1080/15384101.2017.1325039

    Figure Lengend Snippet: SGT1-HSP90 complex is required for composition of the CUL4A complex and recognition of CENP-A by COPS8. (A) SGT1A interacts with CUL4A in vivo. Immunoblot analysis of CUL4A immunoprecipitates. Plasmids pTRM4-SGT1A-Flag and pcDNA3-myc3-CUL4A (Table S3) were cotransfected into HeLa Tet-Off cells. Forty-eight hours after transfection, protein extracts were prepared. Proteins in 1% of the total cell lysate (Input) and precipitate (IP) obtained by using anti-c-myc antibody (Table S1) were detected by Western blot analysis using anti-Flag antibody. (B) Interaction between CENP-A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of endogenous CENP-A immunoprecipitates. Indicated siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa cells. Seventy-two hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using rabbit polyclonal anti-CENP-A antibody (Table S1) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (C) Interaction between CUL4A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of CUL4A immunoprecipitates. Plasmid pTRM4-CUL4A-Flag (Table S3) and indicated siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa Tet-Off cells. Seventy-two hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using ANTI-FLAG M2 Affinity Gel (SIGMAALDRICH) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input). (D) Interaction between CUL4A and COPS8 is reduced in response to SGT1 or HSP90 depletion. Immunoblot analysis of COPS8 immunoprecipitates. The indicated plasmids (Table S3) and siRNA(s) (SGT1 #1 + #2; HSP90 #1; Luc; Table S2) were transfected into HeLa Tet-Off cells. Forty-eight hours after transfection, protein extracts were prepared. Proteins in 3% of the total cell lysate (Input) and precipitate (IP) obtained by using Anti-HA.11 Epitope Tag Affinity Matrix (Covance) were detected by Western blot analysis using the indicated antibodies. GAPDH protein was the loading control (Input).

    Article Snippet: To study the interaction of exogenous CUL4A-Flag with endogenous COPS8 , HeLa Tet-Off cells were cotransfected with plasmids pTRM4-CUL4A-Flag and pCGN-HA-Ubiquitin (Table S3; see Cell culture and transfection).

    Techniques: In Vivo, Transfection, Western Blot, Plasmid Preparation

    CDT2 regulates CHK1 turnover in response to replication stress. (A and B) HU-induced CHK1 turnover in CDT2-depleted cells. HeLa cells transfected with control siRNA (GL3) or siRNA specific for CDT2 were cultured for 48 h and then incubated in medium containing 5 mM HU in the absence or presence of 10 μM MG132. At the indicated times post-HU treatment, cells were harvested and analyzed by Western blotting. A representative experiment is shown in panel A, and the quantitation of data from 5 independent experiments is shown in panel B. (C and D) UV-induced CHK1 turnover in CDT2-depleted cells. HeLa cells transfected with control siRNA or siRNA specific for CDT2 were exposed to 50 J/m 2 UV at 48 h posttransfection. Cells were then cultured in the absence or presence of 10 μM MG132. At the indicated times post-UV exposure, cells were harvested and analyzed by Western blotting. A representative experiment is shown in panel C, and the quantitation of data from 4 independent experiments is shown in panel D. (E) Prolonged half-life of CHK1 in CDT2-depleted cells. HeLa Tet-on cells were transfected with a plasmid encoding Tet-inducible Flag-CHK1 followed by either control siRNA or siRNA specific for CDT2. Cells were cultured in the presence of 0.2 μg/ml doxycycline for 14 h to induce Flag-CHK1 expression. Cells were washed, cultured for 4 h in fresh medium, and then cultured in medium containing 5 mM HU. At the indicated times post-HU treatment, cells were harvested and analyzed by Western blotting. Levels of Flag-CHK1 are shown relative to levels at the 0-h time points (lanes 1 and 4). (F) Effects of CDT1 overexpression on CHK1 stability. HeLa cells expressing Flag-tagged CDT1 for 24 h were incubated in the absence or presence of 5 mM HU for 6 h. Cell lysates were analyzed by Western blotting. The arrowhead indicates endogenous CDT1.

    Journal: Molecular and Cellular Biology

    Article Title: CRL4CDT2 Targets CHK1 for PCNA-Independent Destruction

    doi: 10.1128/MCB.00847-12

    Figure Lengend Snippet: CDT2 regulates CHK1 turnover in response to replication stress. (A and B) HU-induced CHK1 turnover in CDT2-depleted cells. HeLa cells transfected with control siRNA (GL3) or siRNA specific for CDT2 were cultured for 48 h and then incubated in medium containing 5 mM HU in the absence or presence of 10 μM MG132. At the indicated times post-HU treatment, cells were harvested and analyzed by Western blotting. A representative experiment is shown in panel A, and the quantitation of data from 5 independent experiments is shown in panel B. (C and D) UV-induced CHK1 turnover in CDT2-depleted cells. HeLa cells transfected with control siRNA or siRNA specific for CDT2 were exposed to 50 J/m 2 UV at 48 h posttransfection. Cells were then cultured in the absence or presence of 10 μM MG132. At the indicated times post-UV exposure, cells were harvested and analyzed by Western blotting. A representative experiment is shown in panel C, and the quantitation of data from 4 independent experiments is shown in panel D. (E) Prolonged half-life of CHK1 in CDT2-depleted cells. HeLa Tet-on cells were transfected with a plasmid encoding Tet-inducible Flag-CHK1 followed by either control siRNA or siRNA specific for CDT2. Cells were cultured in the presence of 0.2 μg/ml doxycycline for 14 h to induce Flag-CHK1 expression. Cells were washed, cultured for 4 h in fresh medium, and then cultured in medium containing 5 mM HU. At the indicated times post-HU treatment, cells were harvested and analyzed by Western blotting. Levels of Flag-CHK1 are shown relative to levels at the 0-h time points (lanes 1 and 4). (F) Effects of CDT1 overexpression on CHK1 stability. HeLa cells expressing Flag-tagged CDT1 for 24 h were incubated in the absence or presence of 5 mM HU for 6 h. Cell lysates were analyzed by Western blotting. The arrowhead indicates endogenous CDT1.

    Article Snippet: HeLa Tet-on cells (Clontech) were grown in DMEM supplemented with 10% Tet system-approved fetal bovine serum (Clontech), l -glutamine, penicillin-streptomycin, and 100 μg/ml Geneticin (Life Technologies).

    Techniques: Transfection, Cell Culture, Incubation, Western Blot, Quantitation Assay, Plasmid Preparation, Expressing, Over Expression