rabbit anti sumo2 3  (Proteintech)

Journal: mBio

Article Title: EBNA1 SUMOylation by PIAS1 suppresses EBV lytic replication and enhances episome maintenance

doi: 10.1128/mbio.02639-25

Figure Lengend Snippet: PIAS1 enhances EBNA1 SUMOylation both in vivo and in vitro . ( A ) HEK-293T cells were transfected with plasmids encoding Halo-V5-EBNA1, Halo-PIAS1, and His-SUMO2. Whole-cell lysates (input) were analyzed by WB using antibodies against SUMO2/3, PIAS1, V5, and β-actin. SUMOylated proteins are indicated by brackets. EBNA1 was immunoprecipitated using anti-V5 magnetic beads, followed by WB analysis with antibodies as indicated. Arrows denote SUMOylated EBNA1. ( B ) In vitro SUMOylation assay was conducted using a combination of purified proteins, including E1, E2, SUMO2, PIAS1, and the substrate V5-EBNA1, as specified. The reaction was stopped by adding 2× SDS-PAGE loading buffer, followed by WB analysis using anti-V5-HRP antibody. SUMOylated EBNA1 is indicated by brackets. ( C ) WB analysis of PIAS1 and β-actin expression in non-targeting control (NC) and PIAS1-depleted (sg-PIAS1) Akata (EBV+) cells. ( D ) Control (NC) and PIAS1-depleted (sg-PIAS1) Akata (EBV+) cells were blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h at room temperature, followed by incubation with mouse anti-EBNA1 and rabbit anti-SUMO2/3 antibodies. PLA probes were subsequently added for ligation and amplification. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and visualized using a Nikon AXR confocal microscope. The close proximity between EBNA1 and SUMO2/3 is indicated by red fluorescent PLA signals.

Article Snippet: Briefly, cells were blocked with 3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) at room temperature for 1 h, then incubated with PBS control or a mixture of mouse anti-EBNA1 (Cat. #sc-81581, Santa Cruz) and rabbit anti-PIAS1 (Cat. #ab77231, Abcam) or rabbit anti-SUMO2/3 (Cat. # 11251-1-AP, Proteintech) antibodies (1:50 dilution in 3% BSA) at 4°C overnight.

Techniques: In Vivo, In Vitro, Transfection, Immunoprecipitation, Magnetic Beads, Purification, SDS Page, Expressing, Control, Saline, Incubation, Ligation, Amplification, Staining, Microscopy

Journal: bioRxiv

Article Title: DNMT3A Stability Is Maintained by Ubiquitin-Specific Peptidase 11 (USP11) and Sumoylation Countering Degradation

doi: 10.1101/2025.03.05.641683

Figure Lengend Snippet: A, Schematic of immunoprecipitation (IP) mass spectrometry to identify comparing DNMT3A stable (DNMT3A WT or W297Del in the absence of DCAF8) or an unstable protein (W297Del) interactome. 293T cells were engineered to constitutively overexpress the indicated bicistronic GFP-DNMT3A WT or W297Del reporter in the presence or absence of DCAF8. To subjects cell lysate was performed I P and mass spectrometry analysis. B, C, The graph depicts the enriched score of the interaction proteins with stable DNMT3A or unstable DNMT3A. D, 293T cells were transfected with FLAG-DNMT3A1, Myc-USP11 and HA-SUMO3. Cell lysates were immunoprecipitated with anti-FLAG (DNMT3A) antibody, following the detection of DNMT3A1 SUMOylation with anti-HA antibody. E, 293T cells were transfected with FLAG-DNMT3A1, Myc-USP11. Cell lysates were immunoprecipitated with anti-FLAG (DNMT3A) antibody, following the detection of DNMT3A1 endogenous SUMOylation with anti-SUMO2/3 antibody. F. The scheme depicts TAK-981, a first-in-class Inhibitor of SUMO-Activating enzymes. G,H, Kelly or THP-cells were cultured with 1uM ofTAK-981 for 24 hours. Cell lysates were detected with anti-DNMT3A,-SUMO2/3 and -B-actin antibodies.

Article Snippet: Western blotting was performed with anti-FLAG M2-Peroxidase (Sigma, Catalog Number A8592), anti-Myc 9E10 (Santa Cruz Biotechnology, sc-40), anti-HA-Peroxidase (Roche, 12CA5), anti-GFP (NOVUS, Catalog Number NB600-308), anti-USP11 (Abcom, Catalog Number ab109232), anti-Lamin B1 (Santa Cruz Biotechnology, Catalog Number B1304), anti-GAPDH (Cell Signaling Technology, Catalog Number 5174), anti-AIF (Cell Signaling Technology, Catalog Number 5318), Anti-DCAF8 Bethyl lab, Catalog Number A301-556A), anti-B-actin (Cell Signaling Technology, Catalog Number, 3700S), and anti-SUMO2/3 (Cell Signaling Technology, Catalog Number, 4971).

Techniques: Immunoprecipitation, Mass Spectrometry, Transfection, Cell Culture