Journal: Journal of Dental Sciences

Article Title: Rho GTPase activating protein 11A promotes tongue squamous cell carcinoma proliferation and is a transcriptional target of forkhead box M1

doi: 10.1016/j.jds.2024.02.015

Figure Lengend Snippet: ARHGAP11A knockdown induces G1 phase arrest by regulating multiple cell-cycle proteins in tongue squamous cell carcinoma. A-B. Representative fluorescent ubiquitination-based cell cycle indicator (FUCCI) images and ARHGAP11A staining (A) and quantitation of ARHGAP11A mRNA and protein expression in different phases with the cell-cycle (B) in U2OS cells in the human protein atlas (HPA). Image credit: the HPA, data were retrieved from: https://www.proteinatlas.org/ENSG00000198826-ARHGAP11A/subcellular#cell_cycle . C. Western blotting analysis was performed to check the expression of indicated proteins in SCC4 and SCC25 cells 48 h after lentivirus-mediated ARHGAP11A knockdown. D-E. Representative images (D) and quantitation (E) of cell-cycle distribution of SCC4 and SCC25 cells with or without lentivirus-mediated ARHGAP11A knockdown. ∗∗∗, P < 0.001.

Article Snippet: Our review of fluorescent ubiquitination-based cell cycle indicator (FUCCI) staining data from the Human Protein Atlas (HPA) revealed that cells in the G2/M phase (Geminin-positive) exhibited stronger ARHGAP11A staining compared to those in the G1 phase (CDT1-positive) ( A).

Techniques: Knockdown, Staining, Quantitation Assay, Expressing, Western Blot

Journal: Biochimica et Biophysica Acta. Molecular Cell Research

Article Title: A microarray of ubiquitylated proteins for profiling deubiquitylase activity reveals the critical roles of both chain and substrate ☆

doi: 10.1016/j.bbamcr.2012.05.006

Figure Lengend Snippet: DUBs examined for behavior against ubiquitylated substrates were active enzymes. A. The fourteen DUBs examined in Fig. 2 all showed activity (signal versus time over 1 h) by CHOP reporter assay (LifeSensors, Inc.), except AMSH. B. AMSH was examined for activity (versus time over 1 h) against different substrates, di-ubiquitin substrates labeled with an internally quenched fluorophore pair. As expected, activity was specific to K63-linkage of the diubiquitin. C. E3Lite assay data (endpoint RFU signal) for triplicate wells either ubiquitylated with E3 mix (Praja1, Carp2, Murf1) and then mock treated with DUB (“No DUB”); mock ubiquitylated using reaction mixture lacking an E3 and then mock treated with DUB (“Not UB'd”); or ubiquitylated with E3 mix and then treated with DUB enzyme (as listed).

Article Snippet: Since this DUB is known to prefer K63 linked chains, we incubated it with a K63 linked internally quenched fluorescence based di-ubiquitin substrate (LifeSensors, Inc.).

Techniques: Activity Assay, Reporter Assay, Ubiquitin Proteomics, Labeling

Journal: The Journal of Biological Chemistry

Article Title: UbMES and UbFluor: Novel probes for ring-between-ring (RBR) E3 ubiquitin ligase PARKIN

doi: 10.1074/jbc.M116.773200

Figure Lengend Snippet: Chemical activation of the C terminus of ubiquitin as a thioester (e.g. UbMES or UbFluor) can bypass the need for E1, E2, and ATP, downsizing the 5-component native cascade reaction (E1, E2, ATP, PARKIN, and Ub) to 2 components (PARKIN and ByS probes). Transthiolation with UbMES releases a mercaptoethanesulfonate group (MES), whereas transthiolation with UbFluor releases fluorescein thiol.

Article Snippet: UbcH7∼FUb discharge assay UbcH7∼FUb was prepared by incubating Ube1 (0.5 μ m ), UbcH7 (10 μ m ), fluorescent ubiquitin (FUb, LifeSensors, 15 μ m ), and 2 m m ATP in 300 m m NaCl, 8 m m MgCl 2 , 50 m m Tris, pH 7.6, for 30 min at room temperature.

Techniques: Activation Assay, Ubiquitin Proteomics