Journal: Oncogenesis

Article Title: The E3 ubiquitin ligase MG53 inhibits hepatocellular carcinoma by targeting RAC1 signaling

doi: 10.1038/s41389-022-00414-6

Figure Lengend Snippet: A HUH7 cells were transfected with Myc-MG53 plasmid or its mock control. The transfected cells were cultured for 24 h before being further incubated with cycloheximide (CHX) (10 μg/ml) for the indicated time points. The levels of RAC1 at different time points were detected by western blot. Band intensities were quantitatively analyzed and the values were normalized to β-actin. B HUH7 cells were transfected with Myc-MG53 plasmid or its mock control. The transfected cells were cultured for 24 h followed with treatment with MG132 (100 nM) for 6 h. Western blot was performed to detect the protein level of RAC1. The relative RAC1 protein level was statistically analyzed from three independent experiments (right panel). C HUH7 cells were co-transfected with Flag-MG53 and HA-UB plasmid, and protein was denatured before the ubiquitination assay of RAC1. D HUH7 cells were co-transfected with sh-MG53 (or sh-NC) and HA-UB plasmid, and the protein samples were harvested and denatured for subsequent IP assay of ubiquitination. E The MG53 and RAC1 proteins obtained by in vitro protein translation assay were mixed with E1, E2 and UB for in vitro ubiquitination assay. F Recombinant RAC1 loaded with GTP, GDP, or the Mg 2+ -free form (EDTA) were used in the ubiquitination reaction, and the ubiquitination of different forms of RAC1 by MG53 were further analyzed. G HUH7 cells were transfected with Flag-RAC1plasmid, together with Myc-MG53 plasmid or its RING domain deleted mutant plasmids. The transfected cells were treated with MG132 (100 nM) and the ubiquitination of RAC1 was performed by immunoprecipitation assay after the protein samples were denatured. H HUH7 cells were transfected with Flag-MG53 plasmid, together with HA-UB-K48 or HA-UB-K63 plasmid. The transfected cells were treated with MG132 (100 nM) and the protein lysates were harvested and denatured for ubiquitination assay of RAC1. I HUH7 cells were co-transfected with Myc-MG53 and Flag-RAC1 mutants (Flag-RAC1-K5R, Flag-RAC1-K16R, Flag-RAC1-K96R, Flag-RAC1-K116R and Flag-RAC1-K116R), and the transfected cells were treated with MG132 (100 nM). The ubiquitination of RAC1 and its mutants were further analyzed after all of the protein samples were denatured. * P < 0.05, ** P < 0.01 and *** P < 0.001 for statistical analysis of the indicated groups.

Article Snippet: Cycloheximide (CHX) was purchased from ApexBio technology company (Houston, Texas, USA).

Techniques: Transfection, Plasmid Preparation, Control, Cell Culture, Incubation, Western Blot, Ubiquitin Proteomics, In Vitro, Recombinant, Mutagenesis, Immunoprecipitation

Journal: Nature communications

Article Title: Ubiquitin-Binding Site 2 of ataxin-3 prevents its proteasomal degradation by interacting with Rad23

doi: 10.1038/ncomms5638

Figure Lengend Snippet: A) Schematic of ataxin-3. The catalytic triad (red) and two ubiquitin-binding sites (UbS1, light blue and UbS2, orange) are on the catalytic domain (blue). Next are three ubiquitin-interacting motifs (UIMs; red), separated by the polyglutamine (polyQ) region. Preceding the polyQ is the VCP-binding site (green). The NMR-based structure of the catalytic domain of ataxin-3 was reported in ref. . The file for the structure depicted here was obtained from NCBI (PDB: 1YZB) and was rendered and annotated using the software application MacPyMOL. Legend: abbreviations used in this report. B) Western blot of whole cell lysates. HeLa cells were transfected with the indicated constructs and treated with MG132 (6 hours, 15µM) 24 hours post transfection to enhance the capture of ataxin-3 ubiquitination, as we have described before , . We previously showed through stringent immunopurification protocols and mass spectrometry that the more slowly migrating band of ataxin-3 is ubiquitinated ataxin-3 , , , , . Equal total protein loaded (50µg/lane). Transfections were performed independently in triplicate. Overexposed blot to highlight ubiquitinated ataxin-3. C) Top: Western blots of whole cell lysates. HeLa cells were transfected as indicated and treated for the specified amounts of time with cycloheximide (CHX) 48 hours post-transfection. Bottom: Means of ataxin-3 signal quantified from blots on the top and other similar experiments. P values are from Student T-tests comparing protein levels of K-Null ataxin-3 to the wild type counterpart. Error bars: standard deviations. N=6 independently conducted experiments. D) Top: Western blots of whole cell lysates of HeLa cells transfected as indicated, treated or not for 2 hours with the proteasome inhibitor MG132 (10µM) 24 hours after transfection, then treated for the specified amounts of time with cycloheximide (CHX). Bottom: Means of ataxin-3 signal quantified from western blots on the top and other similar, independent experiments. P values are from Student T-tests comparing protein levels of K-Null ataxin-3 to the normal version of the protein. Error bars: standard deviations. N=6 independently conducted experiments.

Article Snippet: Cycloheximide (CHX) was purchased from A.G. Scientific, dissolved in ultra-pure water and used at final concentration of 50 μg/ml.

Techniques: Ubiquitin Proteomics, Binding Assay, Software, Western Blot, Transfection, Construct, Immu-Puri, Mass Spectrometry

Journal: iScience

Article Title: pSNAP: Proteome-wide analysis of elongating nascent polypeptide chains

doi: 10.1016/j.isci.2022.104516

Figure Lengend Snippet:

Article Snippet: cycloheximide (CHX) , FUJIFILM Wako , Cat# 037-20991.

Techniques: Recombinant, Functional Assay, Multiplex sample analysis, Staining, Protease Inhibitor, Lysis, Gene Expression, Membrane, Magnetic Beads, TaqMan Assay, Software

Journal: Chemical Genomics

Article Title: Development of a Yeast Two-Hybrid Screen for Selection of Human Ras-Raf Protein Interaction Inhibitors

doi: 10.1007/978-1-59259-948-6_18

Figure Lengend Snippet: Sensitivity of the modified yeast strain SKY197, relative to the unmodified parental strain SKY191, to various compounds. Decreasing concentrations of cycloheximide (CYH), 4-nitroquinoline-oxide (4-NQO), sulfomethuron methyl (SMM), and Zeocin (Zeo) were plated on parental yeast strain SKY191 and the modified strain SKY197, and incubated on YPD (Glu) or YPG/R (Gal/Raff) media. From top to bottom of each plate, and from left to right of each row: 5 µg, 2.5 µg, 1 µg, 0.5 µg, 0.2 µg, and 50 ng of CYH; 2.5 µg, 1.25 µg, 0.5 µg, 0.25 µg, 0.1 µg, and 25 ng of NQO; 100 µg, 50 µg, 20 µg, 10 µg, 4 µg, and 1 µg of SMM; and 100 µg, 50 µg, 20 µg, 10 µg, 4 µg, and 1 µg of Zeo.

Article Snippet: Cycloheximide (CYH), 4-nitroquinoline-oxide (4-NQO), sulfomethuron methyl (SMM), and 5-fluoro-orotic acid (5FOA) (BioMol, PA).

Techniques: Modification, Incubation

Journal: Chemical research in toxicology

Article Title: Aha1 Is an Autonomous Chaperone for SULT1A1

doi: 10.1021/acs.chemrestox.2c00167

Figure Lengend Snippet: SULT1A1 is a client protein of Aha1. (a) HEK293T cells were transfected with Flag-SULT1A1 for 24 h, treated with cycloheximide (CHX) to block de novo protein synthesis, and harvested at different time points. The cells were then lysed, and the lysates were subjected to Western blot analysis for monitoring the time-dependent alterations of SULT1A1 (Flag), HSP90, and Aha1 proteins. (b,c) HEK293T cells with Aha1 being knocked down with shRNAs targeting its 3′-UTR were reconstituted with wild-type Aha1, E67K mutant, and Aha1-Δ20 for 24 h. The cells were then harvested, and lysates were used to monitor the alterations in the expression level of SULT1A1 protein; “Δ” indicates a nonspecific band. (d,e) The data in (b) and (c) were quantified with ImageJ based on band intensities and normalized against that of GAPDH, where the values are displayed relative to those measured for shCtrl cells. The data represent the mean ± S.D. ( n = 3). The p values were calculated using unpaired, two-tailed Student’s t -test: ns, p ≥ 0.05; *, 0.01 ≤ p < 0.05; **, 0.001 ≤ p < 0.01; ***, p < 0.001.

Article Snippet: Cycloheximide (CHX), 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), ganetespib, and AT13387 were purchased from Fisher Scientific.

Techniques: Transfection, Blocking Assay, Western Blot, Mutagenesis, Expressing, Two Tailed Test