cdc20  (Proteintech)

Journal: Science Advances

Article Title: Deubiquitinase USP8 regulates the spindle assembly checkpoint in oocytes

doi: 10.1126/sciadv.aeb2345

Figure Lengend Snippet: ( A ) Protein levels of the MCC were assessed by immunoblots in control and USP8-depleted oocytes. The blots were probed with USP8, BUBR1, CDC20, BUB3, MAD2, and Cofilin antibodies. ( B ) Localization of BUB3 at the prometaphase I stage in control, USP8-KD, and USP8-rescue oocytes. At 3 hours after GVBD, oocytes were fixed and immunostained for BUB3, CREST, and DNA (Hoechst). Scale bar, 10 μm. ( C ) The relative fluorescence intensities of BUB3 to CREST were measured in control ( n = 200, kinetochores), USP8-KD ( n = 200, kinetochores), and USP8-rescue ( n = 200, kinetochores) oocytes. The signal intensity of BUB3 was normalized with that of CREST. ( D ) Protein levels of BUB3 were assessed by immunoblots in control, USP8-KD, and USP8-rescue oocytes. ( E ) Co-IP result showing the USP8 interaction with BUB3 in oocytes. Mouse oocytes were microinjected with USP8-Flag and BUB3-HA cRNA together or USP8-Flag cRNA alone, maintained for a further 4 hours in 200 μM IBMX to allow time for translation. Target proteins were immunoprecipitated using anti-Flag beads and subjected to Western blotting with Flag and HA antibodies. Input oocyte lysates were immunoblotted with anti-HA antibody to determine the expression of BUB3. ( F ) Co-IP result showing the BUB3 interaction with USP8 in oocytes. Mouse oocytes were microinjected with BUB3-HA and USP8-Flag cRNA together or BUB3-HA cRNA alone, maintained for a further 4 hours in 200 μM IBMX to allow time for translation. Target proteins were immunoprecipitated using anti-HA beads and subjected to Western blotting with HA and Flag antibodies. Input oocyte lysates were immunoblotted with anti-Flag antibody to determine the expression of USP8. Data were presented as the mean percentage (means ± SEM) of at least three independent experiments. *** P < 0.001.

Article Snippet: The blots were further blocked in TBST containing 5% low-fat dry milk for 1 hour at room temperature and then incubated with USP8 (1:500; Abclonal, A7031), USP8 (1:500; Proteintech, 67321-1-Ig), Cyclin B1 (1:1000; Cell Signaling Technology, 4135), BUB3 (1:1000; Abcam, ab133699), BUBR1 (1:1000; Abcam, Ab28193), MAD2 (1:1000; Proteintech, 10337-1-AP), CDC20 (1:500; Proteintech, 10252-1-AP), BUB1 (1:1000; Abcam, Ab195268 ), MPS1 (1:500; Proteintech, 10381-1-AP), Flag (1:1000; Sigma-Aldrich, F3165), HA (1:1000; Sigma-Aldrich, H9658), MYC (1:1000; Cell Signaling Technology, 2278), Normal Rabbit IgG (1:1000; Cell Signaling Technology, 2729), or Cofilin (1:5000; Proteintech, 66057-1-Ig) antibodies at 4°C overnight.

Techniques: Western Blot, Control, Fluorescence, Co-Immunoprecipitation Assay, Immunoprecipitation, Expressing

Journal: World Journal of Gastrointestinal Oncology

Article Title: BTNL9 exerts anti-cancer effects by inhibiting CDC20 to induce G2/M arrest in pancreatic cancer

doi: 10.4251/wjgo.v17.i7.108274

Figure Lengend Snippet: Regulation of BTNL9 expression on downstream molecules. A: Reverse transcription quantitative PCR results of cell cycle related genes; B: The protein expression levels of genes with significant differences in reverse transcription quantitative PCR validation were detected by Western blot; C: Correlation analysis of CDC20 and BNTL9 expression in The Cancer Genome Atlas RNA sequencing data of pancreatic cancer; D: The expression of BTNL9 and CDC20 in pancreatic cancer cells with BTNL9 overexpression detected by immunofluorescence (green = green fluorescence protein; red= CDC20). a P < 0.05, b P < 0.01, c P < 0.001. GFP: Green fluorescence protein.

Article Snippet: CDC20 primary antibody (10252-1-AP, Proteintech) and ABflo 594-conjugated goat anti-rabbit IgG (AS039, ABclonal) were used to perform the immunofluorescence assays.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Biomarker Discovery, Western Blot, RNA Sequencing, Over Expression, Immunofluorescence, Fluorescence

Journal: World Journal of Gastrointestinal Oncology

Article Title: BTNL9 exerts anti-cancer effects by inhibiting CDC20 to induce G2/M arrest in pancreatic cancer

doi: 10.4251/wjgo.v17.i7.108274

Figure Lengend Snippet: Effects of BTNL9/CDC20 on proliferation, migration, and cell cycle of pancreatic cancer cells. A: Pancreatic cancer cells were transfected with BTNL9 and CDC20; B: The proliferative ability of four types of cells in the CCK8 assay; C: Transwell assay was used to analyze the migration capacity in four types of cells; D: The cell cycle distribution of cells was measured by flow cytometry. a P < 0.05, b P < 0.01, c P < 0.001.

Article Snippet: CDC20 primary antibody (10252-1-AP, Proteintech) and ABflo 594-conjugated goat anti-rabbit IgG (AS039, ABclonal) were used to perform the immunofluorescence assays.

Techniques: Migration, Transfection, CCK-8 Assay, Transwell Assay, Flow Cytometry