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    TaKaRa human hela cell cdna library
    Fig. 1. Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B) levels increase in <t>HeLa</t> cell after cisplatin-induced DNA damage. Ac- tivation of the proliferating cell nuclear antigen (PCNA) and phosphorylation of H2A.X following cisplatin treatment (A, B). HeLa cells were treated with cisplatin (100 μM) for 12 h. PCNA (A) and γH2A.X (B) were stained with anti-PCNA and anti-pH2AX (S139) (red) antibodies, respectively. DNA was stained with Hoechst 33342 (blue). Cells were observed using a Nikon inverted fluorescence microscope (TE300). Approximately 400 cells were count- ed. White bar, control; Black bar, cisplatin. Scale bar, 10 μm. (C) Mad2B binding to chromatin was increased following cisplatin treatment. Exponen- tially growing HeLa cells were treated with cisplatin (50 μM) for 12 h and the cytoplasmic and chromatin-associated protein fractions were immunob- lotted with antibodies against Mad2B, histone H3, and α-tubulin. (D) Cisplatin (50 μM) treatment increased the expression of Mad2B as determined via Western blotting of whole HeLa cell extracts (n = 3). Quantitation of the data shown in lower panel. Each bar represents the mean ± SD of three independent experiments (*p < 0.05, **p < 0.01 and ***p < 0.001 using a two-tailed t-test).
    Human Hela Cell Cdna Library, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/human+hela+cdna+libraries/Human+HeLa+QUICK-Clone+cDNA/pm37641805-44-11-16
    Average 91 stars, based on 200 article reviews
    human hela cell cdna library - by Bioz Stars, 2026-09
    91/100 stars

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    1) Product Images from "Mad2B forms a complex with Cdc20, Cdc27, Rev3 and Rev1 in response to cisplatin-induced DNA damage."

    Article Title: Mad2B forms a complex with Cdc20, Cdc27, Rev3 and Rev1 in response to cisplatin-induced DNA damage.

    Journal: The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology

    doi: 10.4196/kjpp.2023.27.5.427

    Fig. 1. Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B) levels increase in HeLa cell after cisplatin-induced DNA damage. Ac- tivation of the proliferating cell nuclear antigen (PCNA) and phosphorylation of H2A.X following cisplatin treatment (A, B). HeLa cells were treated with cisplatin (100 μM) for 12 h. PCNA (A) and γH2A.X (B) were stained with anti-PCNA and anti-pH2AX (S139) (red) antibodies, respectively. DNA was stained with Hoechst 33342 (blue). Cells were observed using a Nikon inverted fluorescence microscope (TE300). Approximately 400 cells were count- ed. White bar, control; Black bar, cisplatin. Scale bar, 10 μm. (C) Mad2B binding to chromatin was increased following cisplatin treatment. Exponen- tially growing HeLa cells were treated with cisplatin (50 μM) for 12 h and the cytoplasmic and chromatin-associated protein fractions were immunob- lotted with antibodies against Mad2B, histone H3, and α-tubulin. (D) Cisplatin (50 μM) treatment increased the expression of Mad2B as determined via Western blotting of whole HeLa cell extracts (n = 3). Quantitation of the data shown in lower panel. Each bar represents the mean ± SD of three independent experiments (*p < 0.05, **p < 0.01 and ***p < 0.001 using a two-tailed t-test).
    Figure Legend Snippet: Fig. 1. Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B) levels increase in HeLa cell after cisplatin-induced DNA damage. Ac- tivation of the proliferating cell nuclear antigen (PCNA) and phosphorylation of H2A.X following cisplatin treatment (A, B). HeLa cells were treated with cisplatin (100 μM) for 12 h. PCNA (A) and γH2A.X (B) were stained with anti-PCNA and anti-pH2AX (S139) (red) antibodies, respectively. DNA was stained with Hoechst 33342 (blue). Cells were observed using a Nikon inverted fluorescence microscope (TE300). Approximately 400 cells were count- ed. White bar, control; Black bar, cisplatin. Scale bar, 10 μm. (C) Mad2B binding to chromatin was increased following cisplatin treatment. Exponen- tially growing HeLa cells were treated with cisplatin (50 μM) for 12 h and the cytoplasmic and chromatin-associated protein fractions were immunob- lotted with antibodies against Mad2B, histone H3, and α-tubulin. (D) Cisplatin (50 μM) treatment increased the expression of Mad2B as determined via Western blotting of whole HeLa cell extracts (n = 3). Quantitation of the data shown in lower panel. Each bar represents the mean ± SD of three independent experiments (*p < 0.05, **p < 0.01 and ***p < 0.001 using a two-tailed t-test).

    Techniques Used: Phospho-proteomics, Staining, Fluorescence, Microscopy, Control, Binding Assay, Expressing, Western Blot, Quantitation Assay, Two Tailed Test

    Fig. 2. Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B) binds to cell division cycle (Cdc)-20 following cisplatin ad- dition. (A) HeLa cells were transfected with HA-Mad2B for 48 h. Cdc20 was immunoprecipitated from the transfected cells following cisplatin treatment (50 μM for 12 h). Immunoprecipitates were immunoblotted with antibodies against HA (middle panel), Cdc20 (upper panel), and γ-tubulin (lower panel). (B) HA-Mad2B co-localizes with Cdc20 follow- ing cisplatin addition. HeLa cells were co-transfected with Myc-Cdc20 and HA-Mad2B for 24 h and then treated with cisplatin (50 μM) for 24 h. Cells were stained for HA-Mad2B (green), DNA (blue), and Myc- Cdc20 (red). Scale bar, 10 μm. (C) Glutathione S-transferase (GST)-pull- down assay. Cell lysates were prepared from exponentially growing cells (interphase) treated with cisplatin (50 μM) for 18 h. Purified GST- Mad2B proteins (0.2–1 μM) were added to the cell lysates. Samples were incubated before the addition of glutathione-sepharose beads. Proteins bound to the beads were immunoblotted with the anti-Cdc20 antibody. As a negative control, lysis buffer was used for the pull-down assay (buffer only). As a positive control, total cell lysates (5% input) were used for identifying Cdc20 on the Western blots (WB). This result is representative of three independent experiments. IP, immunoprecipita- tion; HA, hemagglutinin.
    Figure Legend Snippet: Fig. 2. Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B) binds to cell division cycle (Cdc)-20 following cisplatin ad- dition. (A) HeLa cells were transfected with HA-Mad2B for 48 h. Cdc20 was immunoprecipitated from the transfected cells following cisplatin treatment (50 μM for 12 h). Immunoprecipitates were immunoblotted with antibodies against HA (middle panel), Cdc20 (upper panel), and γ-tubulin (lower panel). (B) HA-Mad2B co-localizes with Cdc20 follow- ing cisplatin addition. HeLa cells were co-transfected with Myc-Cdc20 and HA-Mad2B for 24 h and then treated with cisplatin (50 μM) for 24 h. Cells were stained for HA-Mad2B (green), DNA (blue), and Myc- Cdc20 (red). Scale bar, 10 μm. (C) Glutathione S-transferase (GST)-pull- down assay. Cell lysates were prepared from exponentially growing cells (interphase) treated with cisplatin (50 μM) for 18 h. Purified GST- Mad2B proteins (0.2–1 μM) were added to the cell lysates. Samples were incubated before the addition of glutathione-sepharose beads. Proteins bound to the beads were immunoblotted with the anti-Cdc20 antibody. As a negative control, lysis buffer was used for the pull-down assay (buffer only). As a positive control, total cell lysates (5% input) were used for identifying Cdc20 on the Western blots (WB). This result is representative of three independent experiments. IP, immunoprecipita- tion; HA, hemagglutinin.

    Techniques Used: Transfection, Immunoprecipitation, Staining, Pull Down Assay, Purification, Incubation, Negative Control, Lysis, Positive Control, Western Blot

    Fig. 3. Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B) binding to cell division cycle (Cdc)-27 is increased following cisplatin- induced DNA damage. (A) HeLa cells were treated with cisplatin (50 μM) for 18 h. Cdc27 was immunoprecipitated and immunoblotted with the anti- Mad2B (upper panel) and anti-Cdc27 (low panel) antibodies. (B) Cdc27 was immunoprecipitated and immunoblotted with the anti-HA (upper panel), anti-Cdc27 (lower panel), and anti-α-tubulin (middle panel) antibodies. For control immunoprecipitation (IP), Cdc27 antibody was incubated with the lysis buffer alone. Asterisk indicates a non-specific band (IgG heavy chain). (C) HeLa cells were transfected with HA-Mad2B and treated with cisplatin (50 μM) for 18 h. Cells were stained with the anti-HA (green) and anti-Cdc27 (red) antibodies. DNA was stained with Hoechst 33342 (blue). Scale bar, 5 μm. Molecular weight markers (kD) are indicated on the left of each Western blot (WB). HA, hemagglutinin.
    Figure Legend Snippet: Fig. 3. Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B) binding to cell division cycle (Cdc)-27 is increased following cisplatin- induced DNA damage. (A) HeLa cells were treated with cisplatin (50 μM) for 18 h. Cdc27 was immunoprecipitated and immunoblotted with the anti- Mad2B (upper panel) and anti-Cdc27 (low panel) antibodies. (B) Cdc27 was immunoprecipitated and immunoblotted with the anti-HA (upper panel), anti-Cdc27 (lower panel), and anti-α-tubulin (middle panel) antibodies. For control immunoprecipitation (IP), Cdc27 antibody was incubated with the lysis buffer alone. Asterisk indicates a non-specific band (IgG heavy chain). (C) HeLa cells were transfected with HA-Mad2B and treated with cisplatin (50 μM) for 18 h. Cells were stained with the anti-HA (green) and anti-Cdc27 (red) antibodies. DNA was stained with Hoechst 33342 (blue). Scale bar, 5 μm. Molecular weight markers (kD) are indicated on the left of each Western blot (WB). HA, hemagglutinin.

    Techniques Used: Binding Assay, Immunoprecipitation, Control, Incubation, Lysis, Transfection, Staining, Molecular Weight, Western Blot

    Fig. 5. Activity of the anaphase-promoting complex or cyclosome (APC/C) in response to DNA damage with and without mitotic ar- rest deficient 2 like 2 (Mad2L2, also known as Mad2B) via an in vitro ubiquitination assay. HeLa cells were either transfected with the control small interfering RNA (siRNA) oligonucleotide (10 nM) or Mad2B siRNA oligonucleotide (10 nM). Then, 24 h post-transfection, the cells were treated with 50 μM cisplatin for varying time periods (0–12 h). APC/C was immunopurified from the control and Mad2B-depleted cells. As a positive control, APC/C was immunopurified from mitotic HeLa cells. Immunopurified APC/C was then normalized by quantifica- tion of the protein concentration. A ubiquitination mixture containing E1, E2, ubiquitin, ATP, Myc-tagged Cyclin B N-terminal residues 1-90 (Myc-CycB N90) as a substrate, and 1.5 μM of Cdc20 protein were added. Ubiquitinated Cyclin B (CycB N90) was analyzed via Western blotting with an anti-Myc tag antibody and the quantitated data of ubiquitinated CycB is shown in the lower panel. Lane 1, APC/C without Cdc20 addition (negative control); Lanes 2, 3, and 4, transfection with the control siRNA oligonucleotide; Lanes 5, 6, and 7, transfection with Mad2B siRNA oligonucleotide; Lane 8, APC/C from Nocodazole (0.2 μg/ ml for 16 h)-arrested HeLa cells.
    Figure Legend Snippet: Fig. 5. Activity of the anaphase-promoting complex or cyclosome (APC/C) in response to DNA damage with and without mitotic ar- rest deficient 2 like 2 (Mad2L2, also known as Mad2B) via an in vitro ubiquitination assay. HeLa cells were either transfected with the control small interfering RNA (siRNA) oligonucleotide (10 nM) or Mad2B siRNA oligonucleotide (10 nM). Then, 24 h post-transfection, the cells were treated with 50 μM cisplatin for varying time periods (0–12 h). APC/C was immunopurified from the control and Mad2B-depleted cells. As a positive control, APC/C was immunopurified from mitotic HeLa cells. Immunopurified APC/C was then normalized by quantifica- tion of the protein concentration. A ubiquitination mixture containing E1, E2, ubiquitin, ATP, Myc-tagged Cyclin B N-terminal residues 1-90 (Myc-CycB N90) as a substrate, and 1.5 μM of Cdc20 protein were added. Ubiquitinated Cyclin B (CycB N90) was analyzed via Western blotting with an anti-Myc tag antibody and the quantitated data of ubiquitinated CycB is shown in the lower panel. Lane 1, APC/C without Cdc20 addition (negative control); Lanes 2, 3, and 4, transfection with the control siRNA oligonucleotide; Lanes 5, 6, and 7, transfection with Mad2B siRNA oligonucleotide; Lane 8, APC/C from Nocodazole (0.2 μg/ ml for 16 h)-arrested HeLa cells.

    Techniques Used: Activity Assay, In Vitro, Ubiquitin Proteomics, Transfection, Control, Small Interfering RNA, Positive Control, Protein Concentration, Western Blot, Negative Control

    Related Articles

    Expressing:

    Article Title: N 6 -methyladenosine alters RNA structure to regulate binding of a low-complexity protein
    Article Snippet: The blots were probed with METTL3- (15073-1-AP, Proteintech), METTL14- (HPA038002, Sigma), HNRNPG- (sc-14581 and sc-48796, Santa Cruz Biotechnology) or GAPDH- (A00192-40, Genscript) specific primary antibody, followed by rabbit anti-goat IgG-HRP (sc-2768, Santa Cruz Biotechnology) or goat anti-rabbit IgG-HRP (ab97051, Abcam) secondary antibody, and then visualized by enhanced chemoluminescence (RPN2109, GE Healthcare). .. For expression of the N-terminal RNA recognition motif (N-RRM, residues 1–83) and C-terminal RNA binding domain (C-RBD, residues 334–391) of human HNRNPG protein, sequences encoding these HNRNPG domains were amplified by polymerase chain reaction (PCR) from human HeLa cDNA libraries (637203, Clontech) and then subcloned into pGEX-6p-1 expression vectors using BamHI and XhoI restriction sites. .. Plasmid DNA was transformed into Escherichia coli BL21-CodonPlus(DE3)-RP or BL21-CodonPlus(DE3)-RIL cells (Agilent).

    RNA Binding Assay:

    Article Title: N 6 -methyladenosine alters RNA structure to regulate binding of a low-complexity protein
    Article Snippet: The blots were probed with METTL3- (15073-1-AP, Proteintech), METTL14- (HPA038002, Sigma), HNRNPG- (sc-14581 and sc-48796, Santa Cruz Biotechnology) or GAPDH- (A00192-40, Genscript) specific primary antibody, followed by rabbit anti-goat IgG-HRP (sc-2768, Santa Cruz Biotechnology) or goat anti-rabbit IgG-HRP (ab97051, Abcam) secondary antibody, and then visualized by enhanced chemoluminescence (RPN2109, GE Healthcare). .. For expression of the N-terminal RNA recognition motif (N-RRM, residues 1–83) and C-terminal RNA binding domain (C-RBD, residues 334–391) of human HNRNPG protein, sequences encoding these HNRNPG domains were amplified by polymerase chain reaction (PCR) from human HeLa cDNA libraries (637203, Clontech) and then subcloned into pGEX-6p-1 expression vectors using BamHI and XhoI restriction sites. .. Plasmid DNA was transformed into Escherichia coli BL21-CodonPlus(DE3)-RP or BL21-CodonPlus(DE3)-RIL cells (Agilent).

    Amplification:

    Article Title: N 6 -methyladenosine alters RNA structure to regulate binding of a low-complexity protein
    Article Snippet: The blots were probed with METTL3- (15073-1-AP, Proteintech), METTL14- (HPA038002, Sigma), HNRNPG- (sc-14581 and sc-48796, Santa Cruz Biotechnology) or GAPDH- (A00192-40, Genscript) specific primary antibody, followed by rabbit anti-goat IgG-HRP (sc-2768, Santa Cruz Biotechnology) or goat anti-rabbit IgG-HRP (ab97051, Abcam) secondary antibody, and then visualized by enhanced chemoluminescence (RPN2109, GE Healthcare). .. For expression of the N-terminal RNA recognition motif (N-RRM, residues 1–83) and C-terminal RNA binding domain (C-RBD, residues 334–391) of human HNRNPG protein, sequences encoding these HNRNPG domains were amplified by polymerase chain reaction (PCR) from human HeLa cDNA libraries (637203, Clontech) and then subcloned into pGEX-6p-1 expression vectors using BamHI and XhoI restriction sites. .. Plasmid DNA was transformed into Escherichia coli BL21-CodonPlus(DE3)-RP or BL21-CodonPlus(DE3)-RIL cells (Agilent).

    Article Title: Regulation of co-transcriptional pre-mRNA splicing by m 6 A through the low-complexity protein hnRNPG
    Article Snippet: We therefore resorted to overexpress the full-length protein from baculovirus expression system where the full-length protein was secreted to avoid proteolysis. .. The sequence encoding full-length human hnRNPG protein was amplified from human HeLa cDNA libraries (637203, Clontech) and subcloned into the pGEX-6p-1 vector using BamHI and XhoI restriction sites. .. Plasmids encoding the hnRNPG mutants RRMmut, RGG1mut, and RGG2mut were prepared by QuikChange mutagenesis (200524, Agilent) and Gibson assembly (E2611L, New England BioLabs), with the following mutations in the encoded proteins: K9A, F11A, R49A, F51A, and F53A in RRMmut; R110A, R113A, R120A, and R125A in RGG1mut; R369A, R373A, and R377A in RGG2mut.

    Polymerase Chain Reaction:

    Article Title: N 6 -methyladenosine alters RNA structure to regulate binding of a low-complexity protein
    Article Snippet: The blots were probed with METTL3- (15073-1-AP, Proteintech), METTL14- (HPA038002, Sigma), HNRNPG- (sc-14581 and sc-48796, Santa Cruz Biotechnology) or GAPDH- (A00192-40, Genscript) specific primary antibody, followed by rabbit anti-goat IgG-HRP (sc-2768, Santa Cruz Biotechnology) or goat anti-rabbit IgG-HRP (ab97051, Abcam) secondary antibody, and then visualized by enhanced chemoluminescence (RPN2109, GE Healthcare). .. For expression of the N-terminal RNA recognition motif (N-RRM, residues 1–83) and C-terminal RNA binding domain (C-RBD, residues 334–391) of human HNRNPG protein, sequences encoding these HNRNPG domains were amplified by polymerase chain reaction (PCR) from human HeLa cDNA libraries (637203, Clontech) and then subcloned into pGEX-6p-1 expression vectors using BamHI and XhoI restriction sites. .. Plasmid DNA was transformed into Escherichia coli BL21-CodonPlus(DE3)-RP or BL21-CodonPlus(DE3)-RIL cells (Agilent).

    other:

    Article Title: Regulation of co-transcriptional pre-mRNA splicing by m 6 A through the low-complexity protein hnRNPG
    Article Snippet: The cells were fixed on plates with 1% w/v formaldehyde (28906, Thermo) in the culture media for 10 min at room temperature with gently shaking.

    Sequencing:

    Article Title: Regulation of co-transcriptional pre-mRNA splicing by m 6 A through the low-complexity protein hnRNPG
    Article Snippet: We therefore resorted to overexpress the full-length protein from baculovirus expression system where the full-length protein was secreted to avoid proteolysis. .. The sequence encoding full-length human hnRNPG protein was amplified from human HeLa cDNA libraries (637203, Clontech) and subcloned into the pGEX-6p-1 vector using BamHI and XhoI restriction sites. .. Plasmids encoding the hnRNPG mutants RRMmut, RGG1mut, and RGG2mut were prepared by QuikChange mutagenesis (200524, Agilent) and Gibson assembly (E2611L, New England BioLabs), with the following mutations in the encoded proteins: K9A, F11A, R49A, F51A, and F53A in RRMmut; R110A, R113A, R120A, and R125A in RGG1mut; R369A, R373A, and R377A in RGG2mut.

    Plasmid Preparation:

    Article Title: Regulation of co-transcriptional pre-mRNA splicing by m 6 A through the low-complexity protein hnRNPG
    Article Snippet: We therefore resorted to overexpress the full-length protein from baculovirus expression system where the full-length protein was secreted to avoid proteolysis. .. The sequence encoding full-length human hnRNPG protein was amplified from human HeLa cDNA libraries (637203, Clontech) and subcloned into the pGEX-6p-1 vector using BamHI and XhoI restriction sites. .. Plasmids encoding the hnRNPG mutants RRMmut, RGG1mut, and RGG2mut were prepared by QuikChange mutagenesis (200524, Agilent) and Gibson assembly (E2611L, New England BioLabs), with the following mutations in the encoded proteins: K9A, F11A, R49A, F51A, and F53A in RRMmut; R110A, R113A, R120A, and R125A in RGG1mut; R369A, R373A, and R377A in RGG2mut.



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    pact2 hela cell cdna library  (TaKaRa)
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    TaKaRa pact2 hela cell cdna library
    YFV NS5 (RNApol)- human eIF3L interaction in a yeast two - hybrid system. Yeast strain AH109 was co-transformed with pGBKT7-RNApol (or the empty or Lamin C human protein BD vector) and <t>pACT2-eIF3L</t> (or the empty or SV40 large T-antigen AD vector). The specific interaction was identified by the growth of transformants on SD medium (−LEU, -TRP, -HIS) and SD medium (−LEU, -TRP, -HIS, -ADE) with the activation of the reporter genes HIS 3 and ADE 2. C-, negative control; C+, positive control; N, normalized original culture sample; 1–4, 10-fold serial dilutions of sample.
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    Fig. 1. Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B) levels increase in HeLa cell after cisplatin-induced DNA damage. Ac- tivation of the proliferating cell nuclear antigen (PCNA) and phosphorylation of H2A.X following cisplatin treatment (A, B). HeLa cells were treated with cisplatin (100 μM) for 12 h. PCNA (A) and γH2A.X (B) were stained with anti-PCNA and anti-pH2AX (S139) (red) antibodies, respectively. DNA was stained with Hoechst 33342 (blue). Cells were observed using a Nikon inverted fluorescence microscope (TE300). Approximately 400 cells were count- ed. White bar, control; Black bar, cisplatin. Scale bar, 10 μm. (C) Mad2B binding to chromatin was increased following cisplatin treatment. Exponen- tially growing HeLa cells were treated with cisplatin (50 μM) for 12 h and the cytoplasmic and chromatin-associated protein fractions were immunob- lotted with antibodies against Mad2B, histone H3, and α-tubulin. (D) Cisplatin (50 μM) treatment increased the expression of Mad2B as determined via Western blotting of whole HeLa cell extracts (n = 3). Quantitation of the data shown in lower panel. Each bar represents the mean ± SD of three independent experiments (*p < 0.05, **p < 0.01 and ***p < 0.001 using a two-tailed t-test).

    Journal: The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology

    Article Title: Mad2B forms a complex with Cdc20, Cdc27, Rev3 and Rev1 in response to cisplatin-induced DNA damage.

    doi: 10.4196/kjpp.2023.27.5.427

    Figure Lengend Snippet: Fig. 1. Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B) levels increase in HeLa cell after cisplatin-induced DNA damage. Ac- tivation of the proliferating cell nuclear antigen (PCNA) and phosphorylation of H2A.X following cisplatin treatment (A, B). HeLa cells were treated with cisplatin (100 μM) for 12 h. PCNA (A) and γH2A.X (B) were stained with anti-PCNA and anti-pH2AX (S139) (red) antibodies, respectively. DNA was stained with Hoechst 33342 (blue). Cells were observed using a Nikon inverted fluorescence microscope (TE300). Approximately 400 cells were count- ed. White bar, control; Black bar, cisplatin. Scale bar, 10 μm. (C) Mad2B binding to chromatin was increased following cisplatin treatment. Exponen- tially growing HeLa cells were treated with cisplatin (50 μM) for 12 h and the cytoplasmic and chromatin-associated protein fractions were immunob- lotted with antibodies against Mad2B, histone H3, and α-tubulin. (D) Cisplatin (50 μM) treatment increased the expression of Mad2B as determined via Western blotting of whole HeLa cell extracts (n = 3). Quantitation of the data shown in lower panel. Each bar represents the mean ± SD of three independent experiments (*p < 0.05, **p < 0.01 and ***p < 0.001 using a two-tailed t-test).

    Article Snippet: The human Mad2B gene was cloned by PCR amplification from a human HeLa cell cDNA library (Clontech) using sequence-specific primers.

    Techniques: Phospho-proteomics, Staining, Fluorescence, Microscopy, Control, Binding Assay, Expressing, Western Blot, Quantitation Assay, Two Tailed Test

    Fig. 2. Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B) binds to cell division cycle (Cdc)-20 following cisplatin ad- dition. (A) HeLa cells were transfected with HA-Mad2B for 48 h. Cdc20 was immunoprecipitated from the transfected cells following cisplatin treatment (50 μM for 12 h). Immunoprecipitates were immunoblotted with antibodies against HA (middle panel), Cdc20 (upper panel), and γ-tubulin (lower panel). (B) HA-Mad2B co-localizes with Cdc20 follow- ing cisplatin addition. HeLa cells were co-transfected with Myc-Cdc20 and HA-Mad2B for 24 h and then treated with cisplatin (50 μM) for 24 h. Cells were stained for HA-Mad2B (green), DNA (blue), and Myc- Cdc20 (red). Scale bar, 10 μm. (C) Glutathione S-transferase (GST)-pull- down assay. Cell lysates were prepared from exponentially growing cells (interphase) treated with cisplatin (50 μM) for 18 h. Purified GST- Mad2B proteins (0.2–1 μM) were added to the cell lysates. Samples were incubated before the addition of glutathione-sepharose beads. Proteins bound to the beads were immunoblotted with the anti-Cdc20 antibody. As a negative control, lysis buffer was used for the pull-down assay (buffer only). As a positive control, total cell lysates (5% input) were used for identifying Cdc20 on the Western blots (WB). This result is representative of three independent experiments. IP, immunoprecipita- tion; HA, hemagglutinin.

    Journal: The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology

    Article Title: Mad2B forms a complex with Cdc20, Cdc27, Rev3 and Rev1 in response to cisplatin-induced DNA damage.

    doi: 10.4196/kjpp.2023.27.5.427

    Figure Lengend Snippet: Fig. 2. Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B) binds to cell division cycle (Cdc)-20 following cisplatin ad- dition. (A) HeLa cells were transfected with HA-Mad2B for 48 h. Cdc20 was immunoprecipitated from the transfected cells following cisplatin treatment (50 μM for 12 h). Immunoprecipitates were immunoblotted with antibodies against HA (middle panel), Cdc20 (upper panel), and γ-tubulin (lower panel). (B) HA-Mad2B co-localizes with Cdc20 follow- ing cisplatin addition. HeLa cells were co-transfected with Myc-Cdc20 and HA-Mad2B for 24 h and then treated with cisplatin (50 μM) for 24 h. Cells were stained for HA-Mad2B (green), DNA (blue), and Myc- Cdc20 (red). Scale bar, 10 μm. (C) Glutathione S-transferase (GST)-pull- down assay. Cell lysates were prepared from exponentially growing cells (interphase) treated with cisplatin (50 μM) for 18 h. Purified GST- Mad2B proteins (0.2–1 μM) were added to the cell lysates. Samples were incubated before the addition of glutathione-sepharose beads. Proteins bound to the beads were immunoblotted with the anti-Cdc20 antibody. As a negative control, lysis buffer was used for the pull-down assay (buffer only). As a positive control, total cell lysates (5% input) were used for identifying Cdc20 on the Western blots (WB). This result is representative of three independent experiments. IP, immunoprecipita- tion; HA, hemagglutinin.

    Article Snippet: The human Mad2B gene was cloned by PCR amplification from a human HeLa cell cDNA library (Clontech) using sequence-specific primers.

    Techniques: Transfection, Immunoprecipitation, Staining, Pull Down Assay, Purification, Incubation, Negative Control, Lysis, Positive Control, Western Blot

    Fig. 3. Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B) binding to cell division cycle (Cdc)-27 is increased following cisplatin- induced DNA damage. (A) HeLa cells were treated with cisplatin (50 μM) for 18 h. Cdc27 was immunoprecipitated and immunoblotted with the anti- Mad2B (upper panel) and anti-Cdc27 (low panel) antibodies. (B) Cdc27 was immunoprecipitated and immunoblotted with the anti-HA (upper panel), anti-Cdc27 (lower panel), and anti-α-tubulin (middle panel) antibodies. For control immunoprecipitation (IP), Cdc27 antibody was incubated with the lysis buffer alone. Asterisk indicates a non-specific band (IgG heavy chain). (C) HeLa cells were transfected with HA-Mad2B and treated with cisplatin (50 μM) for 18 h. Cells were stained with the anti-HA (green) and anti-Cdc27 (red) antibodies. DNA was stained with Hoechst 33342 (blue). Scale bar, 5 μm. Molecular weight markers (kD) are indicated on the left of each Western blot (WB). HA, hemagglutinin.

    Journal: The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology

    Article Title: Mad2B forms a complex with Cdc20, Cdc27, Rev3 and Rev1 in response to cisplatin-induced DNA damage.

    doi: 10.4196/kjpp.2023.27.5.427

    Figure Lengend Snippet: Fig. 3. Mitotic arrest deficient 2 like 2 (Mad2L2, also known as Mad2B) binding to cell division cycle (Cdc)-27 is increased following cisplatin- induced DNA damage. (A) HeLa cells were treated with cisplatin (50 μM) for 18 h. Cdc27 was immunoprecipitated and immunoblotted with the anti- Mad2B (upper panel) and anti-Cdc27 (low panel) antibodies. (B) Cdc27 was immunoprecipitated and immunoblotted with the anti-HA (upper panel), anti-Cdc27 (lower panel), and anti-α-tubulin (middle panel) antibodies. For control immunoprecipitation (IP), Cdc27 antibody was incubated with the lysis buffer alone. Asterisk indicates a non-specific band (IgG heavy chain). (C) HeLa cells were transfected with HA-Mad2B and treated with cisplatin (50 μM) for 18 h. Cells were stained with the anti-HA (green) and anti-Cdc27 (red) antibodies. DNA was stained with Hoechst 33342 (blue). Scale bar, 5 μm. Molecular weight markers (kD) are indicated on the left of each Western blot (WB). HA, hemagglutinin.

    Article Snippet: The human Mad2B gene was cloned by PCR amplification from a human HeLa cell cDNA library (Clontech) using sequence-specific primers.

    Techniques: Binding Assay, Immunoprecipitation, Control, Incubation, Lysis, Transfection, Staining, Molecular Weight, Western Blot

    Fig. 5. Activity of the anaphase-promoting complex or cyclosome (APC/C) in response to DNA damage with and without mitotic ar- rest deficient 2 like 2 (Mad2L2, also known as Mad2B) via an in vitro ubiquitination assay. HeLa cells were either transfected with the control small interfering RNA (siRNA) oligonucleotide (10 nM) or Mad2B siRNA oligonucleotide (10 nM). Then, 24 h post-transfection, the cells were treated with 50 μM cisplatin for varying time periods (0–12 h). APC/C was immunopurified from the control and Mad2B-depleted cells. As a positive control, APC/C was immunopurified from mitotic HeLa cells. Immunopurified APC/C was then normalized by quantifica- tion of the protein concentration. A ubiquitination mixture containing E1, E2, ubiquitin, ATP, Myc-tagged Cyclin B N-terminal residues 1-90 (Myc-CycB N90) as a substrate, and 1.5 μM of Cdc20 protein were added. Ubiquitinated Cyclin B (CycB N90) was analyzed via Western blotting with an anti-Myc tag antibody and the quantitated data of ubiquitinated CycB is shown in the lower panel. Lane 1, APC/C without Cdc20 addition (negative control); Lanes 2, 3, and 4, transfection with the control siRNA oligonucleotide; Lanes 5, 6, and 7, transfection with Mad2B siRNA oligonucleotide; Lane 8, APC/C from Nocodazole (0.2 μg/ ml for 16 h)-arrested HeLa cells.

    Journal: The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology

    Article Title: Mad2B forms a complex with Cdc20, Cdc27, Rev3 and Rev1 in response to cisplatin-induced DNA damage.

    doi: 10.4196/kjpp.2023.27.5.427

    Figure Lengend Snippet: Fig. 5. Activity of the anaphase-promoting complex or cyclosome (APC/C) in response to DNA damage with and without mitotic ar- rest deficient 2 like 2 (Mad2L2, also known as Mad2B) via an in vitro ubiquitination assay. HeLa cells were either transfected with the control small interfering RNA (siRNA) oligonucleotide (10 nM) or Mad2B siRNA oligonucleotide (10 nM). Then, 24 h post-transfection, the cells were treated with 50 μM cisplatin for varying time periods (0–12 h). APC/C was immunopurified from the control and Mad2B-depleted cells. As a positive control, APC/C was immunopurified from mitotic HeLa cells. Immunopurified APC/C was then normalized by quantifica- tion of the protein concentration. A ubiquitination mixture containing E1, E2, ubiquitin, ATP, Myc-tagged Cyclin B N-terminal residues 1-90 (Myc-CycB N90) as a substrate, and 1.5 μM of Cdc20 protein were added. Ubiquitinated Cyclin B (CycB N90) was analyzed via Western blotting with an anti-Myc tag antibody and the quantitated data of ubiquitinated CycB is shown in the lower panel. Lane 1, APC/C without Cdc20 addition (negative control); Lanes 2, 3, and 4, transfection with the control siRNA oligonucleotide; Lanes 5, 6, and 7, transfection with Mad2B siRNA oligonucleotide; Lane 8, APC/C from Nocodazole (0.2 μg/ ml for 16 h)-arrested HeLa cells.

    Article Snippet: The human Mad2B gene was cloned by PCR amplification from a human HeLa cell cDNA library (Clontech) using sequence-specific primers.

    Techniques: Activity Assay, In Vitro, Ubiquitin Proteomics, Transfection, Control, Small Interfering RNA, Positive Control, Protein Concentration, Western Blot, Negative Control

    YFV NS5 (RNApol)- human eIF3L interaction in a yeast two - hybrid system. Yeast strain AH109 was co-transformed with pGBKT7-RNApol (or the empty or Lamin C human protein BD vector) and pACT2-eIF3L (or the empty or SV40 large T-antigen AD vector). The specific interaction was identified by the growth of transformants on SD medium (−LEU, -TRP, -HIS) and SD medium (−LEU, -TRP, -HIS, -ADE) with the activation of the reporter genes HIS 3 and ADE 2. C-, negative control; C+, positive control; N, normalized original culture sample; 1–4, 10-fold serial dilutions of sample.

    Journal: Virology Journal

    Article Title: The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication

    doi: 10.1186/1743-422X-10-205

    Figure Lengend Snippet: YFV NS5 (RNApol)- human eIF3L interaction in a yeast two - hybrid system. Yeast strain AH109 was co-transformed with pGBKT7-RNApol (or the empty or Lamin C human protein BD vector) and pACT2-eIF3L (or the empty or SV40 large T-antigen AD vector). The specific interaction was identified by the growth of transformants on SD medium (−LEU, -TRP, -HIS) and SD medium (−LEU, -TRP, -HIS, -ADE) with the activation of the reporter genes HIS 3 and ADE 2. C-, negative control; C+, positive control; N, normalized original culture sample; 1–4, 10-fold serial dilutions of sample.

    Article Snippet: The bait plasmid pGBKT7-RNApol and the pACT2 HeLa cell cDNA library (Clontech, USA) were sequentially introduced into the yeast cells using the EZ-Yeast Transformation kit - Single Vector Transformation protocol (Q.BIO gene, USA).

    Techniques: Transformation Assay, Plasmid Preparation, Activation Assay, Negative Control, Positive Control

    Determination of the YFV NS5 domain responsible for the interaction with eIF3L. The figure shows a representation of RNApol NS5 YFV and its fragments, with deletions (1–14) used to map the interaction domain with the eIF3L protein in the yeast two-hybrid system. The yeast was co-transformed with the pGBKT7-deletion constructs and pACT2-eIF3L. The interaction occurs between amino acids 368 and 448 (interaction domain, the region in black in the schematic representation), which was identified by the growth of transformants on SD medium (−LEU, -TRP, -HIS) and SD medium (−LEU, -TRP, -HIS, -ADE) with the activation of the reporter genes HIS 3 and ADE 2. C-, negative control; C+, positive control; N, normalized original culture sample; 1–4, 10-fold serial dilutions of sample.

    Journal: Virology Journal

    Article Title: The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication

    doi: 10.1186/1743-422X-10-205

    Figure Lengend Snippet: Determination of the YFV NS5 domain responsible for the interaction with eIF3L. The figure shows a representation of RNApol NS5 YFV and its fragments, with deletions (1–14) used to map the interaction domain with the eIF3L protein in the yeast two-hybrid system. The yeast was co-transformed with the pGBKT7-deletion constructs and pACT2-eIF3L. The interaction occurs between amino acids 368 and 448 (interaction domain, the region in black in the schematic representation), which was identified by the growth of transformants on SD medium (−LEU, -TRP, -HIS) and SD medium (−LEU, -TRP, -HIS, -ADE) with the activation of the reporter genes HIS 3 and ADE 2. C-, negative control; C+, positive control; N, normalized original culture sample; 1–4, 10-fold serial dilutions of sample.

    Article Snippet: The bait plasmid pGBKT7-RNApol and the pACT2 HeLa cell cDNA library (Clontech, USA) were sequentially introduced into the yeast cells using the EZ-Yeast Transformation kit - Single Vector Transformation protocol (Q.BIO gene, USA).

    Techniques: Transformation Assay, Construct, Activation Assay, Negative Control, Positive Control

    The eIF3L protein specifically interacts with the N - terminal region of the NS5 YFV interaction domain. The yeast was co-transformed with the pGBKT7 segments and pACT2-eIF3L (or the empty or large T-antigen SV40 AD vector). Segment 3 NS5 (ID) and eIF3L interaction was detected by activation of the reporter genes HIS 3 and ADE 2, which led to the growth of transformants on SD medium (−LEU, -TRP, -HIS) and SD medium (−LEU, -TRP, -HIS, -ADE). C-, negative control; C+, positive control; N, normalized original culture sample; 1–4, 10-fold serial dilutions of sample.

    Journal: Virology Journal

    Article Title: The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication

    doi: 10.1186/1743-422X-10-205

    Figure Lengend Snippet: The eIF3L protein specifically interacts with the N - terminal region of the NS5 YFV interaction domain. The yeast was co-transformed with the pGBKT7 segments and pACT2-eIF3L (or the empty or large T-antigen SV40 AD vector). Segment 3 NS5 (ID) and eIF3L interaction was detected by activation of the reporter genes HIS 3 and ADE 2, which led to the growth of transformants on SD medium (−LEU, -TRP, -HIS) and SD medium (−LEU, -TRP, -HIS, -ADE). C-, negative control; C+, positive control; N, normalized original culture sample; 1–4, 10-fold serial dilutions of sample.

    Article Snippet: The bait plasmid pGBKT7-RNApol and the pACT2 HeLa cell cDNA library (Clontech, USA) were sequentially introduced into the yeast cells using the EZ-Yeast Transformation kit - Single Vector Transformation protocol (Q.BIO gene, USA).

    Techniques: Transformation Assay, Plasmid Preparation, Activation Assay, Negative Control, Positive Control

    The mapping of the terminal region residues of the interaction domain using the yeast two - hybrid system indicates a critical interaction with eIF3L. The yeast was co-transformed with the mutant pGBKT7 constructs and pACT2-eIF3L. A positive interaction is shown by the activation of the reporter gene HIS3 in the interaction of eIF3L with the mutants ID (D436N), ID (D436S), and ID (R439A/H442A), indicating that the mutations are not critical for the interaction. A negative interaction between mutant ID (F431A/W432A/V435A) and eIF3L is indicated by the absence of growth on plates lacking histidine (−His). C-, negative control; C+, positive control; N, normalized original culture sample; 1–4, 10-fold serial dilutions of sample.

    Journal: Virology Journal

    Article Title: The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication

    doi: 10.1186/1743-422X-10-205

    Figure Lengend Snippet: The mapping of the terminal region residues of the interaction domain using the yeast two - hybrid system indicates a critical interaction with eIF3L. The yeast was co-transformed with the mutant pGBKT7 constructs and pACT2-eIF3L. A positive interaction is shown by the activation of the reporter gene HIS3 in the interaction of eIF3L with the mutants ID (D436N), ID (D436S), and ID (R439A/H442A), indicating that the mutations are not critical for the interaction. A negative interaction between mutant ID (F431A/W432A/V435A) and eIF3L is indicated by the absence of growth on plates lacking histidine (−His). C-, negative control; C+, positive control; N, normalized original culture sample; 1–4, 10-fold serial dilutions of sample.

    Article Snippet: The bait plasmid pGBKT7-RNApol and the pACT2 HeLa cell cDNA library (Clontech, USA) were sequentially introduced into the yeast cells using the EZ-Yeast Transformation kit - Single Vector Transformation protocol (Q.BIO gene, USA).

    Techniques: Transformation Assay, Mutagenesis, Construct, Activation Assay, Negative Control, Positive Control

    The interaction domains of other flaviviruses interact with eIF3L. The yeast cells were co-transformed with pGBKT7-ID ( Flavivirus members dengue virus types 3 and 4 and St. Louis encephalitis) and pACT2-eIF3L (or the empty or large T-antigen SV40 AD vector). The interaction domains of other flaviviruses (DENV types 3 and 4 and SLE) present a positive interaction with the eIF3L protein through the activation of the reporter genes HIS 3 and ADE 2. C-, negative control; C+, positive control; N, normalized original culture sample; 1–4, 10-fold serial dilutions of sample.

    Journal: Virology Journal

    Article Title: The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication

    doi: 10.1186/1743-422X-10-205

    Figure Lengend Snippet: The interaction domains of other flaviviruses interact with eIF3L. The yeast cells were co-transformed with pGBKT7-ID ( Flavivirus members dengue virus types 3 and 4 and St. Louis encephalitis) and pACT2-eIF3L (or the empty or large T-antigen SV40 AD vector). The interaction domains of other flaviviruses (DENV types 3 and 4 and SLE) present a positive interaction with the eIF3L protein through the activation of the reporter genes HIS 3 and ADE 2. C-, negative control; C+, positive control; N, normalized original culture sample; 1–4, 10-fold serial dilutions of sample.

    Article Snippet: The bait plasmid pGBKT7-RNApol and the pACT2 HeLa cell cDNA library (Clontech, USA) were sequentially introduced into the yeast cells using the EZ-Yeast Transformation kit - Single Vector Transformation protocol (Q.BIO gene, USA).

    Techniques: Transformation Assay, Plasmid Preparation, Activation Assay, Negative Control, Positive Control