Mol. Cells 2016; 39(11): 834-840
Published online November 18, 2016
https://doi.org/10.14348/molcells.2016.0238
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: yshim@konkuk.ac.kr
Keywords
Cdc25 phosphatase promotes cell cycle through dephosphorylation of cyclin-dependent kinases (Cdks) (Fantes, 1979). There are four
Nematodes were cultured and handled at 20°C using standard methods (Brenner, 1974).
To examine the number of intestinal nuclei marked by GFP using an
RNAi depletion of
HeLa cells were maintained in Dulbecco’s Modified Eagle’s Medium (Welgene, Korea), supplemented with 10% fetal bovine serum (Welgene), 100 U/ml penicillin (Welgene), and 100 μg/mL streptomycin (Welgene) in a humidified atmosphere with 5% CO2 at 37°C.
To generate Myc-tagged CDC-25.1 and CDC-25.2 fusion proteins at their N-terminal, cDNA sequences of
Cell pellets were resuspended in NP-40 lysis buffer (150 mM NaCl, 1.0% NP-40, 50 mM Tris-Cl, pH 8.0) containing protease inhibitor mixture (Roche Applied Bioscience, Germany) and incubated for 30 min at 4°C, and sonicated (CosmoBio, Japan). Then, 650 μg of cell lysates were incubated with protein G-sepharose (Sigma, USA) at 4°C overnight, and 2 μg of monoclonal anti-c-Myc antibody (9E10, Santa Cruz, USA) was added to the samples. The samples were incubated for 4 h at 4°C with gentle rotation. After washing with NP-40 lysis buffer 4 times, samples were boiled in sodium dodecyl sulfate (SDS) loading buffer for 10 min. Boiled cell lysate proteins were separated by 7–10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, USA) using a semidry transfer apparatus (Hoefer, USA). The membrane was blocked with 5% non-fat dried milk/2% BSA in TBS-T (150 mM NaCl, 50 mM Tris-Cl, 0.1% Tween 20) for 30 min at RT. The blots on the membrane were first incubated with the following primary antibodies at 4°C overnight: anti-HA antibody (1:1,000, Y-11, Santa Cruz); anti-Myc antibody (1:1,000, 9E10, Santa Cruz); anti-GFP antibody (1:1,000, A11122, Invitrogen, USA); monoclonal anti-FLAG M2 antibody (1:500, F1804, Sigma); monoclonal anti-α-Tubulin antibody (1:5,000, T9026, Sigma). The blots on the membrane were then incubated with the secondary antibody, horseradish peroxidase-conjugated anti-mouse IgG (1:5,000, Santa Cruz) or horseradish peroxidase-conjugated anti-rabbit IgG (1:5,000, Santa Cruz), at RT for 2 h. Finally, immunoreactive blots were detected as chemiluminescence signals by using ECL reagent (Amersham® Biotechnology, UK) and a Bio-imaging Analyzer LAS-4000 (Fuji-film, Japan).
HeLa cells were co-transfected with plasmid DNAs expressing HA-tagged ubiquitin, Myc-tagged CDC-25.1 or CDC-25.2, and GFP-tagged LIN-23 for 48 h and then treated with 25 μM of MG132 (Sigma) for 6 h to inhibit the activity of proteasome and then harvested. The harvested cell pellets were then lysed in the NP-40 lysis buffer with protease inhibitors as previously described. Myc-tagged CDC-25.1 or Myc-tagged CDC-25.2 was immunoprecipitated with anti-Myc antibody (9E10, Santa Cruz) and protein G sepharose beads (Sigma). The immunoprecipitates were washed four times with NP-40 lysis buffer and boiled in SDS loading buffer for 10 min. The immunoprecipitated and boiled samples were then analyzed by Western blot analysis.
The bar graphs are shown as the average ± S.D. Box-and-whisker plots are displayed using Tukey whiskers, extending up to data points 1.5 interquartile ranges away from the first and the third quartile.
In a previous genome-wide RNAi screen of ubiquitination-related genes, RNAi depletion of 17 genes induced excess intestinal cell divisions during the embryogenesis (Du et al., 2015). Among the 17 genes,
In
To examine whether LIN-23, a
To examine whether the observed physical interaction between LIN-23 and CDC-25.1 and that between LIN-23 and CDC-25.2 actually increased the ubiquitination of CDC-25.1 and CDC-25.2, respectively, an
LIN-23 is a
A previous bioinformatics study revealed 866 potential ubiquitination-related genes in
Mutant alleles of C. elegans E3 ubiquitin ligase-component genes examined in this study
Gene | Allele | Class of E3 ligase complex | Reference |
---|---|---|---|
lin-23 | e1883 | CRL1/SCF (F-box) | Kipreos et al., 2000 |
cul-1 | e1756 | CRL1/SCF (Cullin) | Kipreos et al., 1996 |
cul-2 | or209 | CRL2 (Cullin) | Burger et al., 2013 |
skr-1 | ok1696 | CRL1/SCF (Skp1) | Nayak et al., 2002 |
skr-2 | ok1938 | CRL1/SCF (Skp1) | Nayak et al., 2002 |
rbx-1 | ok782 | CRL1, 2, 3 | Kamura et al., 1999 |
zyg-11 | b2 | CRL2 (VHL) | Sonneville and Gönczy, 2004 |
zif-1 | gk117 | CRL2 (SOCS-box) | DeRenzo et al., 2003 |
mel-26 | or184 | CRL2 (BTB) | Pintard et al., 2003 |
mat-2 | ax76 | APC/C | Golden et al., 2000 |
mat-3 | ku233 | APC/C | Golden et al., 2000 |
rnf-113 | ok1401 | RING-type | Lee et al., 2013 |
rbpl-1 | ok907 | RING-type | Huang et al., 2013 |
rfp-1 | ok572 | RING-type | Crowe and Candido, 2004 |
Mol. Cells 2016; 39(11): 834-840
Published online November 30, 2016 https://doi.org/10.14348/molcells.2016.0238
Copyright © The Korean Society for Molecular and Cellular Biology.
Miseol Son1, Ichiro Kawasaki1, Bong-Kyeong Oh2, and Yhong-Hee Shim1,*
1Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Korea, 2Institute of Medical Science, Hanyang University College of Medicine, Seoul 04763, Korea
Correspondence to:*Correspondence: yshim@konkuk.ac.kr
Keywords:
Cdc25 phosphatase promotes cell cycle through dephosphorylation of cyclin-dependent kinases (Cdks) (Fantes, 1979). There are four
Nematodes were cultured and handled at 20°C using standard methods (Brenner, 1974).
To examine the number of intestinal nuclei marked by GFP using an
RNAi depletion of
HeLa cells were maintained in Dulbecco’s Modified Eagle’s Medium (Welgene, Korea), supplemented with 10% fetal bovine serum (Welgene), 100 U/ml penicillin (Welgene), and 100 μg/mL streptomycin (Welgene) in a humidified atmosphere with 5% CO2 at 37°C.
To generate Myc-tagged CDC-25.1 and CDC-25.2 fusion proteins at their N-terminal, cDNA sequences of
Cell pellets were resuspended in NP-40 lysis buffer (150 mM NaCl, 1.0% NP-40, 50 mM Tris-Cl, pH 8.0) containing protease inhibitor mixture (Roche Applied Bioscience, Germany) and incubated for 30 min at 4°C, and sonicated (CosmoBio, Japan). Then, 650 μg of cell lysates were incubated with protein G-sepharose (Sigma, USA) at 4°C overnight, and 2 μg of monoclonal anti-c-Myc antibody (9E10, Santa Cruz, USA) was added to the samples. The samples were incubated for 4 h at 4°C with gentle rotation. After washing with NP-40 lysis buffer 4 times, samples were boiled in sodium dodecyl sulfate (SDS) loading buffer for 10 min. Boiled cell lysate proteins were separated by 7–10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, USA) using a semidry transfer apparatus (Hoefer, USA). The membrane was blocked with 5% non-fat dried milk/2% BSA in TBS-T (150 mM NaCl, 50 mM Tris-Cl, 0.1% Tween 20) for 30 min at RT. The blots on the membrane were first incubated with the following primary antibodies at 4°C overnight: anti-HA antibody (1:1,000, Y-11, Santa Cruz); anti-Myc antibody (1:1,000, 9E10, Santa Cruz); anti-GFP antibody (1:1,000, A11122, Invitrogen, USA); monoclonal anti-FLAG M2 antibody (1:500, F1804, Sigma); monoclonal anti-α-Tubulin antibody (1:5,000, T9026, Sigma). The blots on the membrane were then incubated with the secondary antibody, horseradish peroxidase-conjugated anti-mouse IgG (1:5,000, Santa Cruz) or horseradish peroxidase-conjugated anti-rabbit IgG (1:5,000, Santa Cruz), at RT for 2 h. Finally, immunoreactive blots were detected as chemiluminescence signals by using ECL reagent (Amersham® Biotechnology, UK) and a Bio-imaging Analyzer LAS-4000 (Fuji-film, Japan).
HeLa cells were co-transfected with plasmid DNAs expressing HA-tagged ubiquitin, Myc-tagged CDC-25.1 or CDC-25.2, and GFP-tagged LIN-23 for 48 h and then treated with 25 μM of MG132 (Sigma) for 6 h to inhibit the activity of proteasome and then harvested. The harvested cell pellets were then lysed in the NP-40 lysis buffer with protease inhibitors as previously described. Myc-tagged CDC-25.1 or Myc-tagged CDC-25.2 was immunoprecipitated with anti-Myc antibody (9E10, Santa Cruz) and protein G sepharose beads (Sigma). The immunoprecipitates were washed four times with NP-40 lysis buffer and boiled in SDS loading buffer for 10 min. The immunoprecipitated and boiled samples were then analyzed by Western blot analysis.
The bar graphs are shown as the average ± S.D. Box-and-whisker plots are displayed using Tukey whiskers, extending up to data points 1.5 interquartile ranges away from the first and the third quartile.
In a previous genome-wide RNAi screen of ubiquitination-related genes, RNAi depletion of 17 genes induced excess intestinal cell divisions during the embryogenesis (Du et al., 2015). Among the 17 genes,
In
To examine whether LIN-23, a
To examine whether the observed physical interaction between LIN-23 and CDC-25.1 and that between LIN-23 and CDC-25.2 actually increased the ubiquitination of CDC-25.1 and CDC-25.2, respectively, an
LIN-23 is a
A previous bioinformatics study revealed 866 potential ubiquitination-related genes in
. Mutant alleles of C. elegans E3 ubiquitin ligase-component genes examined in this study.
Gene | Allele | Class of E3 ligase complex | Reference |
---|---|---|---|
lin-23 | e1883 | CRL1/SCF (F-box) | Kipreos et al., 2000 |
cul-1 | e1756 | CRL1/SCF (Cullin) | Kipreos et al., 1996 |
cul-2 | or209 | CRL2 (Cullin) | Burger et al., 2013 |
skr-1 | ok1696 | CRL1/SCF (Skp1) | Nayak et al., 2002 |
skr-2 | ok1938 | CRL1/SCF (Skp1) | Nayak et al., 2002 |
rbx-1 | ok782 | CRL1, 2, 3 | Kamura et al., 1999 |
zyg-11 | b2 | CRL2 (VHL) | Sonneville and Gönczy, 2004 |
zif-1 | gk117 | CRL2 (SOCS-box) | DeRenzo et al., 2003 |
mel-26 | or184 | CRL2 (BTB) | Pintard et al., 2003 |
mat-2 | ax76 | APC/C | Golden et al., 2000 |
mat-3 | ku233 | APC/C | Golden et al., 2000 |
rnf-113 | ok1401 | RING-type | Lee et al., 2013 |
rbpl-1 | ok907 | RING-type | Huang et al., 2013 |
rfp-1 | ok572 | RING-type | Crowe and Candido, 2004 |
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