Journal: PLoS Pathogens

Article Title: Multiple UBXN family members inhibit retrovirus and lentivirus production and canonical NFκΒ signaling by stabilizing IκBα

doi: 10.1371/journal.ppat.1006187

Figure Lengend Snippet: (A) FLAG-UBXN1 was co-transfected with Myc-Cul1, as indicated, and immunoprecipitated (IP) using anti-FLAG antibody, followed by immunoblotting (IB) using anti-FLAG or Myc antibody; β−tubulin served as loading control; (B) Left: schematic of Cul1 deletion constructs, with indicated regions that bind to Skp1 and Rbx1 (green and yellow, respectively, not precisely to scale); right: FLAG-Cul1 constructs were transfected into HEK293 cells, as indicated at top; endogenous UBXN1 was detected using anti-UBXN1 antibody and β−tubulin again served as loading control; (C) left: At top: Co-IP of endogenous UBXN1 and Cul1 from HFFs, after stimulating cells with 10 ng/mL TNFα; input of various proteins shown at bottom; right: relative band intensity of IκBα and Cul1, normalized against the density of β−tubulin (in the left immunoblot image); (D) left: Transfection of HEK293 cells with either empty vector or plasmid encoding FLAG-UBXN1, treated with 5 ng/mL TNFα for the indicated times, and cell lysates immunoblotted for all three indicated proteins, with β−tubulin serving as loading control; right: relative band intensity of IκBα normalized against the density of β−tubulin (in the left immunoblot image); (E) Similar to (D) except that NFκB-FFLUC reporter was co-transfected and cell lysates assayed for luciferase activity, normalized to co-transfected Renilla-LUC reporter, in the presence (grey bars) or absence (black bars) of UBXN1. Experiments of (C) and (D) were repeated at least twice, with similar results.

Article Snippet: Rabbit anti-Cul1 monoclonal antibody was from Novus Biologicals (NBP1-40523).

Techniques: Transfection, Immunoprecipitation, Western Blot, Control, Construct, Co-Immunoprecipitation Assay, Plasmid Preparation, Luciferase, Activity Assay

Journal: PLoS Pathogens

Article Title: Multiple UBXN family members inhibit retrovirus and lentivirus production and canonical NFκΒ signaling by stabilizing IκBα

doi: 10.1371/journal.ppat.1006187

Figure Lengend Snippet: (A) 293T cells were transiently transfected with the indicated FLAG-UBXN1 constructs or FLAG-Cul1 in the presence of either Myc-Rbx1 or Myc-Skp1 as indicated, with co-IP at top and input, including β-tubulin, at bottom; (B) Similar to (A) , except decreasing amounts of Myc-UBXN1 were transfected along with Myc-Skp1 and either empty vector (EV), ½-1 FLAG-Cul1, FLAG-Cul1, or 1/3-1 FLAG-Cul1, as indicated, with co-IP at top and input, including β-tubulin, at bottom; (C) Similar to (B) , except that Myc-Rbx1 was transfected along with EV, either 0-1/2 FLAG-Cul1, FLAG-Cul1, or 0-2/3 FLAG-Cul1, as indicated, with co-IP at top and input, including Cox IV, at bottom. Note that co-expression of Cul1 increased Rbx1 levels.

Article Snippet: Rabbit anti-Cul1 monoclonal antibody was from Novus Biologicals (NBP1-40523).

Techniques: Transfection, Construct, Co-Immunoprecipitation Assay, Plasmid Preparation, Expressing

Journal: PLoS Pathogens

Article Title: Multiple UBXN family members inhibit retrovirus and lentivirus production and canonical NFκΒ signaling by stabilizing IκBα

doi: 10.1371/journal.ppat.1006187

Figure Lengend Snippet: (A) Left: immunoblot of indicated proteins from HFFs stably transduced with either empty HIV-based vector or vector encoding anti-UBXN1 shRNA, after stimulation with 5 ng/mL TNFα for indicated times; right: relative band intensity of IκBα, normalized to β−tubulin (in the left immunoblot image); (B) Quantification of NFκB (left) and HIV LTR (right) FFLUC reporters in cell lines of (A) , normalized to co-transfected Renilla-LUC plasmid; for NFκB reporter cells were treated for 4 h with 5 ng/mL TNFα 48 h post-transfection; (C) Left: immunoblot of indicated proteins from UBXN1-/- HPRT-/- and control HPRT-/- MEFs, after stimulation with 10 ng/mL TNFα for indicated times (upper) or treated with 1 μM Bortezomib for 5 h and then stimulated with 10 ng/mL TNFα for the indicated times (lower); right: relative band intensity of IκBα, normalized to β−tubulin (in the left immunoblot images); (D) Confocal immunofluorescence microscopy of UBXN1 and Cul1 in UBXN1-/- HPRT-/- and control HPRT-/- MEFs. Nuclear DNA stained with TO-PRO-3 (I); UBXN1 (II) and Cul1 (III) were stained using secondary antibodies conjugated to Alexa Fluor 546 and 488, respectively; IV shows merge; Scale bar = 10 μm; (E) Quantification of NFκB and HIV LTR-FFLUC reporters in UBXN1-/- HPRT-/- and control HPRT-/- MEFs. Left: NFκB-FFLUC values 48 h post-transfection in the presence of 5 ng/mL TNFα for 4h, normalized to Renilla-LUC reporter; right: HIV LTR-FFLUC values 48 hrs post-transfection, similarly normalized; (F) JLAT10.6 T cells stably transduced with either HIV-based vector encoding anti-UBXN1 shRNA or control empty vector, after stimulating the cells with 5 ng/mL TNFα for the indicated times and measuring % eGFP+ by FACS. Data in (B) and (E) represent mean ± SEM (n = 3). ** p < 0.005, * p < 0.05 by student’s t-test. Experiments of (A) and (C) were repeated several times, with similar results.

Article Snippet: Rabbit anti-Cul1 monoclonal antibody was from Novus Biologicals (NBP1-40523).

Techniques: Western Blot, Stable Transfection, Transduction, Plasmid Preparation, shRNA, Transfection, Control, Immunofluorescence, Microscopy, Staining

Journal: PLoS Pathogens

Article Title: Multiple UBXN family members inhibit retrovirus and lentivirus production and canonical NFκΒ signaling by stabilizing IκBα

doi: 10.1371/journal.ppat.1006187

Figure Lengend Snippet: (A) Cartoon rendering of how UBXN1 may be interfering with Cul1 in the canonical NFκB signaling pathway; (B) and (C) additional schematics of how UBXN1 may somehow be interfering with Cul1 activity either by steric hindrance (B) or allosteric effect (C) . UBXN1 is known to multimerize and interacts with both N and C termini of Cul1; stoichiometry between UBXN1 and Cul1 is not known (dashed ovals). In both models Skp1 and Rbx1 interact with the N and C termini of Cul1, respectively, and UBXN1 interacts with Rbx1 but not Skp1. These models do not exclude the possibility that another factor or protein (e.g., multimerized ubiquitin) mediates the interaction between UBXN1 and Cul1/Rbx1.

Article Snippet: Rabbit anti-Cul1 monoclonal antibody was from Novus Biologicals (NBP1-40523).

Techniques: Activity Assay, Ubiquitin Proteomics

Journal: Fish Physiology and Biochemistry

Article Title: Thymol and menthol as anaesthetics for short transportation of zebrafish larva

doi: 10.1007/s10695-025-01530-x

Figure Lengend Snippet: a Nrf2 protein levels in 96 hpf zebrafish larvae following 4 h transportation. b Representative Western blots of Nrf2. Data are expressed as mean ± SD for parametric data distribution. Statistical analysis was performed using Student’s t test (unpaired) to compare the naïve group with the control group, and used one-way ANOVA followed by Dunnett’s multiple-comparison test for comparation of the control and anaesthetized groups. Asterisks indicate significative differences between the control and anaesthetized groups ( p < 0.05)

Article Snippet: The primary antibodies for GAPDH (1:1000, GTX100118, 36 kDa, GeneTex) and Nrf2 (1:20, PCRP-NFE2L2–1D12, 68 kDa, DSHB) were used overnight following the blockade of the membrane with 5% BSA.

Techniques: Western Blot, Control, Comparison