prk5 ha ub k48 vector  (Addgene inc)

Journal: Cell Reports Medicine

Article Title: KRAS G12D -driven pentose phosphate pathway remodeling imparts a targetable vulnerability synergizing with MRTX1133 for durable remissions in PDAC

doi: 10.1016/j.xcrm.2025.101966

Figure Lengend Snippet:

Article Snippet: In addition, the cDNA sequences of E2F1 and RING1 gene were cloned into the pRK5-HA vector. pCMV3-His-Rb (#HG10137-NH) was purchased from Sino Biological Inc. (Beijing, China). pRK5-HA-Ubi, pRK5- Ring1 , pRK5- UBE2T , deletions-mutant p53 plasmids were constructed in our previous article.

Techniques: Mutagenesis, Recombinant, Virus, CCK-8 Assay, Membrane, Concentration Assay, Modification, Transfection, Agarose Gel Electrophoresis, Plasmid Preparation, Extraction, Bicinchoninic Acid Protein Assay, Staining, RNA Extraction, H&E Stain, Viability Assay, Reporter Assay, Labeling, Western Blot, Sequencing, Control, Software

Journal: Biomolecules

Article Title: OTUD7B Activates Wnt Signaling Pathway through the Interaction with LEF1

doi: 10.3390/biom13061001

Figure Lengend Snippet: OTUD7B interacts with LEF1 and activates Wnt signaling. ( A ) HEK293T cells were transiently transfected with MYC-LEF1 and SFB-tagged DUBs (USP26, USP36, USP42, and OTUD7B) and subjected to pulldown using S-protein beads. The pulled-down DUBs and DUB-bound LEF1 were detected via immunoblotting using anti-FLAG and anti-MYC antibodies, respectively. ( B ) HEK293T cells were transiently transfected with MYC-LEF1 and SFB-OTUD7B and subjected to immunoprecipitation using MYC antibody-conjugated agarose beads. The pulled-down LEF1 and bound OTUD7B were detected via immunoblotting using anti-MYC and FLAG antibodies, respectively. ( C ) HEK293T cells were co-transfected with TOPflash (LEF1/β-catenin-responsive luciferase vector) or its mutant FOPflash, along with Renilla luciferase and SFB-USP36 or SFB-OTUD7B plasmids. Firefly luciferase activity from TOPflash and FOPflash was normalized to Renilla luciferase activity. The normalized TOPflash luminescence levels were further normalized to each normalized FOPflash luminescence. SFB-GFP was used as a negative control. ( D ) HEK293T cells were co-transfected with TOPflash or FOPflash, along with Renilla luciferase, and control siRNA or siRNA for OTUD7B. Left panel: the cells were subjected to lysis and immunoblotting was conducted using OTUD7B antibody to confirm the knockdown of endogenous OTUD7B. Right panel: Firefly luciferase activity from TOPflash and FOPflash was normalized to Renilla luciferase activity. The normalized TOPflash luminescence levels were further normalized to each normalized FOPflash luminescence. Control siRNA-transfected group was used as a negative control. Error bars represent the standard error of the mean (S.E.M.). Statistical significance was determined using an unpaired t -test.

Article Snippet: HA-ubiquitin, TOPflash, and FOPflash vectors were obtained from Addgene (Item #17608, #12456, and #12457, respectively; Watertown, MA, USA).

Techniques: Transfection, Western Blot, Immunoprecipitation, Luciferase, Plasmid Preparation, Mutagenesis, Activity Assay, Negative Control, Control, Lysis, Knockdown

Journal: PLoS ONE

Article Title: Ubiquitin-Specific Protease 4 Inhibits Mono-Ubiquitination of the Master Growth Factor Signaling Kinase PDK1

doi: 10.1371/journal.pone.0031003

Figure Lengend Snippet: A : HEK293T cells were transfected with 6×His-tagged ubiquitin and ubiquitinated proteins were isolated with nickel beads under denaturing conditions. Pull-downs and input were analyzed with an antibody recognizing PDK1. Ub-PDK1 indicates ubiquitin-modified PDK1 species; asterisk indicates unspecific cross-reacting band. B : HEK293T cells were transfected with a V5-tagged PDK1 cDNA and V5-PDK1 was immunoprecipitated. Immunoprecipitations (IP) and input were immunoblotted with a V5 antibody. C : HEK293T cells were co-transfected as indicated with HA-tagged ubiquitin and V5-PDK1. Immunoprecipitation of HA-ubiquitin and input were immunoblotted with a V5 antibody. D : HEK293T cells were co-transfected as indicated and whole cell extracts were probed with anti-V5 or anti-HA antibody. E : HEK293T cells were transfected with V5-tagged PDK1 or a linear PDK1-ubiquitin C-terminal fusion protein. The whole cell extracts were probed with anti-V5 antibody. F : Endogenous PDK1 was immunoprecipitated from HEK293T cells and blotted with anti-PDK1 antibody. The following controls were used: anti-PDK1 antibody only, sepharose beads only, anti-lgG2a control antibody with beads. G : Lysates from the indicated cell lines were subjected to PDK1 immunoprecipitation. Immunoprecipitations were immunoblotted with a PDK1 antibody.

Article Snippet: HA-tagged ubiquitin expression vector (pRK5-HA-ubiquitin wild type) and the 68 DUB expression vectors were obtained from Addgene. cDNAs encoding the full-length USP4-WT and -C311S proteins in the pcDNA4-MYC-His vectors were kindly provided by Dr. Kristina Lindsten and subcloned into pcDNA6-V5-His.

Techniques: Transfection, Isolation, Modification, Immunoprecipitation