Journal: The Journal of Biological Chemistry

Article Title: Deubiquitinating enzyme USP39 promotes the growth and metastasis of gastric cancer cells by modulating the degradation of RNA-binding protein RBM39

doi: 10.1016/j.jbc.2024.107751

Figure Lengend Snippet: Proteomic identification of RBM39-interacting proteins. A , schematic illustration of the experimental procedure for the identification of RBM39-interacting proteins. HEK293T cells were transfected with pcDNA3.1 or FLAG-RBM39 plasmid for 48 h and lysed in the modified radioimmunoprecipitation assay buffer. FLAG-RBM39 and its interacting proteins were purified by anti-FLAG affinity gel, separated with SDS-PAGE, reduced with dithiothreitol, alkylated with chloroacetic amide, digested with trypsin, desalted with C18 ziptip, and analyzed with mass spectrometry. B , silver staining of proteins that were purified by anti-FLAG affinity gel and separated with SDS-PAGE. The red arrow indicated FLAG-RBM39. C , the volcano plot of RBM39-interacting proteins obtained from three biological replicates of proteomic analysis of anti-FLAG affinity gel purified samples. -Log 10 ( p value) and Log 2 (intensity RBM39 /intensity pcDNA3.1 ) were obtained from Perseus. The blue dot and red dots represent RBM39 and deubiquitinating enzymes (DUBs), respectively. D – E , biological process and Kyoto Encyclopedia of Genes and Genomes pathway analysis of potential RBM39-interacting proteins using DAVID bioinformatics resources ( https://david.ncifcrf.gov ).

Article Snippet: Proteins were eluted with buffer containing FLAG peptide (200 μg/ml, DYKDDDDK, 20571ES11, Yeasen) or 2×sample loading buffer.

Techniques: Transfection, Plasmid Preparation, Modification, Radio Immunoprecipitation, Purification, SDS Page, Mass Spectrometry, Silver Staining

Journal: The Journal of Biological Chemistry

Article Title: Deubiquitinating enzyme USP39 promotes the growth and metastasis of gastric cancer cells by modulating the degradation of RNA-binding protein RBM39

doi: 10.1016/j.jbc.2024.107751

Figure Lengend Snippet: Biochemical validation of the interaction between USP39 and RBM39. A – B , exogenous expressed USP39 and RBM39 interact with each other. HEK293T cells were transfected with the indicated plasmids for 48 h and USP39 or RBM39-interacting proteins were immunoprecipitated with anti-FLAG affinity gel. Cell lysates and immunoprecipitates were immunoblotted with the indicated antibodies. C – D , exogenously expressed USP39 and RBM39 interact with the endogenous RBM39 and USP39. HEK293T cells were transfected with FLAG-USP39 or HA-RBM39 plasmids for 48 h, followed by anti-FLAG or anti-HA immunoprecipitation and Western blotting with the indicated antibodies. E – F , USP39 interacts with RBM39 endogenously. MKN45 and HGC27 cells were lysed and endogenous USP39 and its interacting proteins were immunoprecipitated with anti-USP39 antibody. Cell lysates and immunoprecipitates were immunoblotted with RBM39 and USP39. IgG was used in the control group. ∗: antibody heavy chain. G , RBM39 and USP39 colocalize in the nucleus. HEK293 cells were transfected with HA-RBM39 and/or FLAG-USP39 plasmids for 48 h, washed with ice-cold PBS, fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100, incubated with FLAG/HA primary antibodies and fluorescent secondary antibodies, stained with DAPI, and photographed under a confocal laser microscope. The scale bar represents 20 μm. Intensity traces from the original images were obtained using the plot profile tool in ImageJ and plotted on the right side . Colocalization coefficient was calculated by Pearson’s correlation analysis. Red : RBM39; green : USP39; blue : DAPI. DAPI, 4′,6-diamidino-2-phenylindole; HA, hemagglutinin.

Article Snippet: Proteins were eluted with buffer containing FLAG peptide (200 μg/ml, DYKDDDDK, 20571ES11, Yeasen) or 2×sample loading buffer.

Techniques: Biomarker Discovery, Transfection, Immunoprecipitation, Western Blot, Control, Incubation, Staining, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Deubiquitinating enzyme USP39 promotes the growth and metastasis of gastric cancer cells by modulating the degradation of RNA-binding protein RBM39

doi: 10.1016/j.jbc.2024.107751

Figure Lengend Snippet: USP39 upregulates the RBM39 protein level through its deubiquitinating activity. A , USP39 expression increased RBM39. AGS cells were transfected with pcDNA3.1 and FLAG-USP39 plasmids, lysed, and immunoblotted with the indicated plasmids. B , USP39 knockdown diminished RBM39. AGS cells transfected with si NC or si USP39 for 48 h were harvested to obtain cell lysates for Western blotting analysis. C , USP39 expression did not alter RBM39 mRNA level. AGS cells were transfected with pcDNA3.1 and FLAG-USP39 plasmids. Proteins and RNAs were extracted with radioimmunoprecipitation assay buffer and TRIzol, respectively. The expression levels of RBM39 protein and mRNA were evaluated by Western blotting and real-time fluorescence quantitative PCR, respectively, after synthesis of the first strand cDNA. D , the deubiquitinating activity was required for USP39 to regulate RBM39. AGS cells were transfected with USP39 and its catalytically inactive C306A mutant, lysed, and the cell lysates were immunoblotted. Mean ± SD (n = 3), Student’s t test with group comparisons test ( A – C ), one-way ANOVA with Dunnett’s multiple comparisons test ( D ), ∗∗: p < 0.01, ∗∗∗: p < 0.001, and ns, not significant. Note: the difference in the fold change of RBM39 in ( A ) and ( D ) upon USP39 expression may be caused by the different expression levels of USP39 in two experiments.

Article Snippet: Proteins were eluted with buffer containing FLAG peptide (200 μg/ml, DYKDDDDK, 20571ES11, Yeasen) or 2×sample loading buffer.

Techniques: Activity Assay, Expressing, Transfection, Knockdown, Western Blot, Radio Immunoprecipitation, Fluorescence, Real-time Polymerase Chain Reaction, Mutagenesis