Journal: Oncotarget

Article Title: βTrCP controls the lysosome-mediated degradation of CDK1, whose accumulation correlates with tumor malignancy

doi:

Figure Lengend Snippet: (A) Five peptides of CDK1, sequences underlined, were observed by MS/MS. (B) Whole cell extracts (WCE) from HCT116 or HCT116 transfected cells were used to immunoprecipitate βTrCP (left panel) or CDK1 (right panel), and complexes were analyzed by immunoblotting. IP-PI: immunoprecipitation with normal rabbit (left panel) or mouse (right panel) sera. (C) HCT116 cells were synchronized in the different phases of the cell cycle as described in Methods, and analyzed by Western blot (left panel) or by immunoprecipitation (right panel). (D, E, F) HeLa, Cos-7 or HCT116 cells were transfected with the indicated plasmids and analyzed by immunoblot. (G) U2OS cells were interfered with the indicated siRNAs and analyzed by Western blot. (H) HeLa cells were interfered with the indicated siRNAs, and 24 hours before harvesting were synchronized using different drugs: butyrate (G1 phase), aphidicolin (APH, S phase), RO-3306 (G2 phase) or nocodazole (NZ, M phase). Extracts were blotted with the indicated antibodies. (I) HCT116 cells were transfected with pFlagCMV2-CDK1 and interfered with the indicated siRNAs, and analyzed by Western blot. (J) In vitro ubiquitin ligation assay of 35 S labeled in vitro -transcribed/translated CDK1 was conducted in the presence or absence of the following products: cold in vitro -transcribed/translated βTrCP, βTrCPΔF or FBXW7α, E1 (His 6 -ubiquitin activating enzyme), E2 (His 6 -UbcH3 and UbcH5a) and Ub (ubiquitin). Samples were incubated at 30°C for 1h. The bracket on the right side marks a ladder of bands corresponding to poly-ubiquitinated CDK1. (K) Cos-7 cells were transfected with the indicated plasmids and poly-ubiquitinated Flag-CDK1 visualized after Western blot of Flag immunoprecipitations. Anti-K 48 -Ub and K 63 -Ub polyclonal antibodies recognize K48- and K63-linkage specific ubiquitination, respectively. The quantitative fold change in CDK1 or Flag-CDK1 was determined relative to the loading control.

Article Snippet: After dewaxing, sections were subjected to heat-induced epitope retrieval in 10mM EDTA pH 9.0, and were incubated with either anti-βTrCP rabbit polyclonal antibody (1:150; Cell Signaling) or anti-CDK1 rabbit polyclonal antibody (1:400; Santa Cruz) overnight at 4oC.

Techniques: Tandem Mass Spectroscopy, Transfection, Western Blot, Immunoprecipitation, In Vitro, Ubiquitin Proteomics, Ligation, Labeling, Incubation, Control

Journal: Oncotarget

Article Title: βTrCP controls the lysosome-mediated degradation of CDK1, whose accumulation correlates with tumor malignancy

doi:

Figure Lengend Snippet: (A) HCT116 cells were treated with proteasomal (LLnL) or/and lysosomal (ConA) inhibitors for 4 hours and protein expression analyzed by immunoblot. (B) HCT116 cells were transfected with the indicated plasmids and treated or not with ConA 4 hours before harvesting. Total extracts were blotted with different antibodies. (C) U2OS cells were interfered and treated with LLnL or ConA 4 hours before harvested. Extracts were analyzed by immunoblot. (D) Comparison of CDK1 ubiquitination from transfected cells treated or not with ConA. Flag-CDK1 was immunoprecipitated and blotted with the indicated antibodies. (E, F) HeLa cells were transfected or interfered and treated with proteasome inhibitors. Extracts were analyzed by Western blot. The quantitative fold change in CDK1 was determined relative to the loading control.

Article Snippet: After dewaxing, sections were subjected to heat-induced epitope retrieval in 10mM EDTA pH 9.0, and were incubated with either anti-βTrCP rabbit polyclonal antibody (1:150; Cell Signaling) or anti-CDK1 rabbit polyclonal antibody (1:400; Santa Cruz) overnight at 4oC.

Techniques: Expressing, Western Blot, Transfection, Comparison, Ubiquitin Proteomics, Immunoprecipitation, Control

Journal: Oncotarget

Article Title: βTrCP controls the lysosome-mediated degradation of CDK1, whose accumulation correlates with tumor malignancy

doi:

Figure Lengend Snippet: (A) Alignment of the putative βTrCP consensus sequence identified in human CDK1 (residues 41 to 46) with other orthologs. Arrows indicate the amino acid changes performed to obtain a CDK1 mutated in this motif (CDK1 βM, CDK1 βTrCP mutant). (B) Cos-7 cells were transfected with the indicated plasmids and extracts analyzed by immunoblot. (C) βTrCP was immunoprecipitated from Cos-7 transfected cells, and coimmunoprecipitation of Flag-CDK1 or Flag-CDK1 βM studied with anti-Flag. Anti-β catenin was used as a control of βTrCP immunoprecipitation. IP-PI: immunoprecipitation with normal rabbit serum. WCE: whole cell extracts. (D) Cos-7 cells were transfected with the indicated plasmids, and Flag-CDK1 or Flag-CDK1 βM ubiquitination obtained after Flag immunoprecipitation and Western blot using anti-ubiquitin. The quantitative fold change in Flag-CDK1 was determined relative to the loading control.

Article Snippet: After dewaxing, sections were subjected to heat-induced epitope retrieval in 10mM EDTA pH 9.0, and were incubated with either anti-βTrCP rabbit polyclonal antibody (1:150; Cell Signaling) or anti-CDK1 rabbit polyclonal antibody (1:400; Santa Cruz) overnight at 4oC.

Techniques: Sequencing, Mutagenesis, Transfection, Western Blot, Immunoprecipitation, Control, Ubiquitin Proteomics

Journal: Oncotarget

Article Title: βTrCP controls the lysosome-mediated degradation of CDK1, whose accumulation correlates with tumor malignancy

doi:

Figure Lengend Snippet: (A) Cell lines were treated with doxorubicin (Dx) for 24 hours, and CDK1 expression analyzed by immunoblot. (B) Protein synthesis of HCT116 cells was arrested with cycloheximide (CHX) and treated or not with Dx for 10 hours. Extracts were analyzed by Western blot. (C) HeLa cells were treated with Dx for 24 hours and with C16, a PKR inhibitor, 2 hours before harvested. Proteins expression was studied with different antibodies. (D) HeLa cells were interfered with the indicated siRNAs, and 24 hours before harvested, treated with Dx. Extracts were blotted with the indicated antibodies. (E) HeLa cells were treated with poly(I:C), a PKR activator, for 6 hours, and extracts were analyzed by immunoblot. p-PKR indicates phospho-PKR (Thr451). (F, G) HEK293T and HeLa cells were treated as in (D). (H) HeLa cells were interfered with the indicated siRNAs and extracts analyzed by immunoblot. (I) LNCaP and PC3 cells were γ-irradiated to a dose of 10 Gy and harvested 4h and 8h later. Extracts were blotted with the indicated antibodies. C: control. (J) LNCaP and PC3 cells were treated as in (A). cl PARP: cleaved PARP. (K) LNCaP cells were transfected with the indicated plasmids and treated or not with Dx for 24 hours before harvested. Extracts were analyzed by Western blot. cl PARP: cleaved PARP. (L) LNCaP cells were treated as in (D). cl PARP: cleaved PARP. The quantitative fold change in CDK1 was determined relative to the loading control.

Article Snippet: After dewaxing, sections were subjected to heat-induced epitope retrieval in 10mM EDTA pH 9.0, and were incubated with either anti-βTrCP rabbit polyclonal antibody (1:150; Cell Signaling) or anti-CDK1 rabbit polyclonal antibody (1:400; Santa Cruz) overnight at 4oC.

Techniques: Expressing, Western Blot, Irradiation, Control, Transfection

Journal: Oncotarget

Article Title: βTrCP controls the lysosome-mediated degradation of CDK1, whose accumulation correlates with tumor malignancy

doi:

Figure Lengend Snippet: (A) Immunohistochemical expression of βTrCP and CDK1 in human breast carcinomas (a-f), gastric carcinomas (g-l), and cholangiocarcinomas (m-r). Bars, 200μm. (B) Immunohistochemical expression of βTrCP and CDK1 in benign prostate glands (a-c), low grade prostatic carcinomas (d-i), and high grade prostatic carcinomas (j-r). HE, hematoxylin-eosin. Bars, 200μm.

Article Snippet: After dewaxing, sections were subjected to heat-induced epitope retrieval in 10mM EDTA pH 9.0, and were incubated with either anti-βTrCP rabbit polyclonal antibody (1:150; Cell Signaling) or anti-CDK1 rabbit polyclonal antibody (1:400; Santa Cruz) overnight at 4oC.

Techniques: Immunohistochemical staining, Expressing

Journal: Oncotarget

Article Title: βTrCP controls the lysosome-mediated degradation of CDK1, whose accumulation correlates with tumor malignancy

doi:

Figure Lengend Snippet: In all cell lines tested, CDK1 (in blue) is ubiquitinated and degraded by SCF βTrCP /lysosome in control conditions (basal phosphorylation status). In highly tumorigenic cell lines, DNA damage inhibits CDK1 degradation (in red), whereas in poorly tumorigenic cell lines it enhances CDK1 degradation (thick black arrow). CDK1 (in purple) indicates both CDK1 from control conditions and after DNA damage. Ub (in black): ubiquitinated CDK1.

Article Snippet: After dewaxing, sections were subjected to heat-induced epitope retrieval in 10mM EDTA pH 9.0, and were incubated with either anti-βTrCP rabbit polyclonal antibody (1:150; Cell Signaling) or anti-CDK1 rabbit polyclonal antibody (1:400; Santa Cruz) overnight at 4oC.

Techniques: Control, Phospho-proteomics

Journal: Endocrinology

Article Title: Silencing Med12 Gene Reduces Proliferation of Human Leiomyoma Cells Mediated via Wnt/ β -Catenin Signaling Pathway

doi: 10.1210/en.2016-1097

Figure Lengend Snippet: Antibody Table

Article Snippet: Cdk1/Cdk2 , Anti-Cdk1 antibody , Santa Cruz Biotechnology (Catalog # sc-53219) , Mouse monoclonal , 500.

Techniques:

Journal: Endocrinology

Article Title: Silencing Med12 Gene Reduces Proliferation of Human Leiomyoma Cells Mediated via Wnt/ β -Catenin Signaling Pathway

doi: 10.1210/en.2016-1097

Figure Lengend Snippet: Effect of Med12 knockdown on cell cycle regulatory and growth-promoting pathways in hUF cells. (A) Equal amounts of cell lysates from Med12-shRNA and control-shRNA cells were analyzed by Western blotting using anti-cyclin D1, Cdk1, Cdk2, Cdk4, and Cdk6 antibodies. β-actin Western blot was used as the loading control. (B) The intensity of each protein band was quantified and normalized with corresponding β-actin. *P < 0.05 when compared with control-shRNA. (C) Cell lysates were analyzed by Western blot using antibodies against p-ERK, total ERK, p-AKT, and total AKT. (D) The intensity of each protein band was quantified and normalized with corresponding β-actin. *P < 0.05 when compared with control shRNA.

Article Snippet: Cdk1/Cdk2 , Anti-Cdk1 antibody , Santa Cruz Biotechnology (Catalog # sc-53219) , Mouse monoclonal , 500.

Techniques: Knockdown, shRNA, Control, Western Blot

Journal: International Journal of Oncology

Article Title: Knockdown of PGBD5 inhibits the malignant progression of glioma through upregulation of the PPAR pathway

doi: 10.3892/ijo.2024.5643

Figure Lengend Snippet: List of antibodies used.

Article Snippet: CDK1 Rabbit mAb , ABclonal Biotech Co., Ltd. (cat. no. A11420).

Techniques:

Journal: International Journal of Oncology

Article Title: Knockdown of PGBD5 inhibits the malignant progression of glioma through upregulation of the PPAR pathway

doi: 10.3892/ijo.2024.5643

Figure Lengend Snippet: Effects of PGBD5 knockdown on the apoptosis of glioma cells. (A and B) Effects of PGBD5 knockdown on the cell cycle progression of glioma cells. (D and E) Apoptosis and cell cycle distribution of glioma cells were detected using flow cytometry. Western blotting showed the expression levels of (C) apoptosis-related proteins (Bax, Bcl-2 and caspase-3) and (F) cell cycle-related proteins (CDK1 and cyclin B1) in glioma cell lines after PGBD5 knockdown. β-actin or GAPDH were used as the protein loading controls. The experiments were repeated three times. * P<0.05, ** P<0.01, *** P<0.001 vs. sh-NC or as indicated. NC, negative control; PGBD5, piggyBac transportable element derived; sh, short hairpin.

Article Snippet: CDK1 Rabbit mAb , ABclonal Biotech Co., Ltd. (cat. no. A11420).

Techniques: Knockdown, Flow Cytometry, Western Blot, Expressing, Negative Control, Derivative Assay

Journal: eLife

Article Title: Stress-activated MAPK signaling controls fission yeast actomyosin ring integrity by modulating formin For3 levels

doi: 10.7554/eLife.57951

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-Cdk1/Cdc2 (PSTAIR)(rabbit polyclonal) , Millipore , Cat#: 06–923; RRID: AB_310302 , WB (1:1000).

Techniques: Reverse Transcription, SYBR Green Assay, Western Blot, Software