ubiquitin rhodamine 110  (BPS Bioscience)

Journal: Journal of microbiology and biotechnology

Article Title: Helicobacter pylori -Induced Progranulin Promotes the Progression of the Gastric Epithelial Cell Cycle by Regulating CDK4.

doi: 10.4014/jmb.2203.03053

Figure Lengend Snippet: Fig. 1. Differences of PGRN and CDK4 protein expression between gastric cancer tissue and adjacent normal tissue. (A, B) Expression levels of PGRN (A) and CDK4 (B) in both gastric cancer and adjacent normal tissues as measured by immunohistochemistry. The results represent the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, paired t-test. All data are mean values of three biological replicates.

Article Snippet: They were then incubated at 4oC overnight after adding mouse anti-PGRN antibody (1:100, sc-377036, Santa Cruz) or rabbit anti-CDK4 antibody (1:800, #12790, Cell Signaling Technology) according to the instructions.

Techniques: Expressing, Immunohistochemistry

Journal: Journal of microbiology and biotechnology

Article Title: Helicobacter pylori -Induced Progranulin Promotes the Progression of the Gastric Epithelial Cell Cycle by Regulating CDK4.

doi: 10.4014/jmb.2203.03053

Figure Lengend Snippet: Fig. 4. PGRN positively regulates CDK4 to promote cell cycle progression. (A, B) qPCR and western blot analysis of the expression of CDK4 in BGC-823 cells infected with H. pylori 26695 at a MOI of 100: 1. (C, D) qPCR and western blot analysis of the expression of CDK4 after transfection with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus. (E, F) CDK4 expression after transfection with CDK4-RNAi-13, CDK4-RNAi-14, CDK4-RNAi-15 of CDK4- knockdown lentivirus. (G) Flow cytometry analysis of the cell cycle changes of CDK4 knockdown and coculture with H. pylori in 50:1 MOI in BGC- 823 cells. (H) Flow cytometry analysis of the cell cycle changes of CDK4 knockdown cell lines infected with PGRN-knockdown /overexpressed lentivirus and cocultured with H. pylori at a MOI of 50: 1. The results represent the mean ± SD of three independent experiments. SI, PGRN knockdown group, NS, the control group of PGRN knockdown, GFP, the control group of PGRN overexpressing, ns, not significant, HP, Helicobacter pylori, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: They were then incubated at 4oC overnight after adding mouse anti-PGRN antibody (1:100, sc-377036, Santa Cruz) or rabbit anti-CDK4 antibody (1:800, #12790, Cell Signaling Technology) according to the instructions.

Techniques: Western Blot, Expressing, Infection, Transfection, shRNA, Knockdown, Flow Cytometry, Control

Journal: Journal of microbiology and biotechnology

Article Title: Helicobacter pylori -Induced Progranulin Promotes the Progression of the Gastric Epithelial Cell Cycle by Regulating CDK4.

doi: 10.4014/jmb.2203.03053

Figure Lengend Snippet: Fig. 5. PGRN regulates CDK4 through the PI3K/Akt signaling pathway and promotes progression of the gastric mucosal epithelial cell cycle. (A) qPCR analysis of the expression of PGRN after transfection with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus and cocultured with H. pylori at a MOI of 100: 1. (B) Western blot analysis of CDK4 protein expression in cells pretreated with a PI3K signal pathway inhibitor (LY294002) for 2 h before transfection with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus. (C) qPCR analysis of the expression of CDK4 after transfection with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus and cocultured with H. pylori at a MOI of 100: 1. (D) Western blot analysis of CDK4, Akt, and p-Akt protein expression in cells transfected with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus and cocultured with H. pylori at a MOI of 100: 1. The results represent the mean ± SD of three independent experiments. SI, PGRN knockdown group, NS, the control group of PGRN knockdown, GFP, the control group of PGRN overexpressing, HP, Helicobacter pylori, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: They were then incubated at 4oC overnight after adding mouse anti-PGRN antibody (1:100, sc-377036, Santa Cruz) or rabbit anti-CDK4 antibody (1:800, #12790, Cell Signaling Technology) according to the instructions.

Techniques: Expressing, Transfection, shRNA, Western Blot, Knockdown, Control

Journal: Molecular cancer research : MCR

Article Title: USP22 interacts with PALB2 and promotes chemotherapy resistance via homologous recombination of DNA double strand breaks

doi: 10.1158/1541-7786.MCR-19-0053

Figure Lengend Snippet: PALB2WD40 directly binds USP22 and stimulates its catalytic DUB activity. a. In-silico model using ZDOCK of USP22DUB binding PALB2WD40 predicting key residues involved in the interaction. b. U2OSFOKI cells co-transfected with the indicated mCherry-LacI protein to tether it to 256X LacO array along with GFP-USP22. Percentage of cells with recruitment of GFP-USP22 to LacO array are indicated with +/− SD. White scale bars indicated 5um. 120 cells were counted for each condition for every experimental round. Experiment was done in triplicate. c. Immunoprecipitation of mcherry-PALB2 fragments plasmids transiently transfected into H1299 cells. Western blot against mCherry, FLAG (FLAG-USP22), Rad51. mCherry alone is used as a negative control. Numbers to the left of western blots indicate molecular weight. d. In-vitro pulldown using recombinant MBP-PALB2WD40 (prey) and His-USP22FL (bait). MBP was also incubated with His-USP22 as a negative control. Western blot was performed against His (His-USP22) and MBP (MBP-PALB2WD40, MBP). Numbers to the left of western blots indicate molecular weight. e. Ubiquitin cleavage assay using rhodamine-ubiquitin that is quenched and upon cleavage of ubiquitin fluoresces. Proteins indicated below were pre-incubated to form their respective complexes then rhodamine-ubiquitin was added. Units on vertical axis indicate raw fluorescent units at 565nm. Buffer alone was used as a negative control for auto-fluorescence baseline. Error bars indicated +/− SD. Experiment was done in triplicate.

Article Snippet: Rhodamine-Ubiquitin (BPS Bioscience, #81151) was then added in at a 1:1 molar ratio and incubated with the complexes at 37°C for 1 hr.

Techniques: Activity Assay, In Silico, Binding Assay, Transfection, Immunoprecipitation, Western Blot, Negative Control, Molecular Weight, In Vitro, Recombinant, Incubation, Cleavage Assay, Fluorescence