Journal: The FASEB Journal

Article Title: The homeostatic function of Regnase‐2 restricts neuroinflammation

doi: 10.1096/fj.202201978r

Figure Lengend Snippet: FIGURE 6 IL-1β induces modification and degradation of Reg-2. U251-MG cells with SB transposon-based Dox-inducible expression of FLAG-tagged wild-type Reg-2 under the control of a promoter containing three tetracycline operator sites (3xTO; A) or seven tetracycline operator sites (7xTO; B–F) were treated with Dox (1 μg/mL) for 24 h. Then, 7xTO cells were pre-treated with cycloheximide (CHX) (100 μg/mL) for 1 h or left untreated and stimulated with IL-β (10 ng/mL) or TNF (10 ng/mL) (B, C) while 3xTO cells were stimulated with IL-1β only (A). (D) The cells were treated with Dox (1 μg/mL) for 24 h and stimulated with IL-1β (10 ng/mL) for 60 min. The protein lysate was incubated with phosphatase λ-PP (60 min, 37°C). (E) The modification of Reg-2 following IL-β stimulation (10 ng/mL, 60 min) with an MG-132 proteasome inhibitor (1 μM) was examined. (F) Immunoassay of lysates of U251-MG cells with Dox-inducible expression of FLAG- tagged wild-type Reg-2 under the control of 7xTO promoter and transfected with the expression vector encoding HA-tagged Ubiquitin. The cells were stimulated for 0–30 min with IL-1β (10 ng/mL), followed by Reg-2 immunoprecipitation (IP) and immunoblot analysis (IB) with antibody to ubiquitin (Ub) or Reg-2 (FLAG). Levels of the indicated proteins were examined by the western blotting. Red asterisks indicate a slower migrating band. Data are representative of three to five independent experiments.

Article Snippet: U251- MG cells modified with pSBtet- GP- Regnase- 2 vector were seeded on 60- mm cell culture dishes and the following day were transfected with a pRK5- HAUbiquitin- WT plasmid encoding HA- tagged Ubiquitin (pRK5- HA- Ubiquitin- WT was a gift from Ted Dawson (Addgene plasmid # 17608))50 or left untreated.

Techniques: Modification, Expressing, Control, Incubation, Transfection, Plasmid Preparation, Ubiquitin Proteomics, Immunoprecipitation, Western Blot

Journal: Molecular cell

Article Title: A Rapid Induction Mechanism for Lin28a in Trophic Responses

doi: 10.1016/j.molcel.2016.12.025

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following plasmids were obtained as generous gifts: Wild-type (WT) TRBP, phospho mutant (SΔA) TRBP, and phospho-mimic (SΔD) TRBP (from Dr. Zain Paroo ( Paroo et al., 2009 )); FLAG-Lin28a (from Dr. Yinqun Huang ( Xu et al., 2009 )); HA-Ubiquitin K48 (from Dr. Ted Dawson ( Lim et al., 2005 )); FL-Lin28bΔNLS mutant (from Dr. Richard Gregory ( Piskounova et al., 2011 )); HA-CAMAPKK (pMCL-HA-MAPKK1-R4F[(Δ31-51)S218E/S222D] from Dr. Natalie Ahn, Addgene plasmid # 40810 ( Mansour et al., 1994 )); HA-Merlin (from Dr. Kunliang Guan, Addgene plasmid # 32836 ( Zhao et al., 2007 )).

Techniques: Recombinant, Protease Inhibitor, TaqMan microRNA Assay, Plasmid Preparation, shRNA, Software

Journal: Journal of Biological Chemistry

Article Title: N-Methyl-D-aspartate Receptor Subtype Mediated Bidirectional Control of p38 Mitogen-activated Protein Kinase

doi: 10.1074/jbc.m502080200

Figure Lengend Snippet: FIG. 6. Inhibition of calcineurin sig- nificantly attenuates p38 activation, but inhibition of nNOS and CaMKII has no effect. Neurons were pretreated for 1 h in the presence of 1 M FK506 or in Me2SO control (vehicle, DMSO). A, with 100 M NMDA treatment, FK506 signifi- cantly inhibited p38 activation at 1 min. *, p 0.0012, by two-tailed Student’s t test; compiled from four experiments. All samples have similar phospho-p38 levels at 5 min. B, representative blots for phos- pho-p38, total p38, and phospho-ERK1/2 show inhibition of phospho-p38 levels af- ter 5 min of 10 M NMDA treatment with 1 M FK506, but no effect was noted on total p38 or phospho-ERK1/2 levels. C, 1 M FK506 inhibited p38 activation when compared with 10 M NMDA treatment with Me2SO control, at both the 1-min (#, p 0.0041) and 5-min time points (†, p 0.0176, by two-tailed Student’s t test, compiled from four experiments). No sig- nificant differences were noted between FK506 and Me2SO pretreatments with- out NMDA treatment. D, 1 M FK506 () pretreatment did not affect intracellular calcium transients with 10 M NMDA (Œ), as noted by calcium imaging, compiled from three experiments containing 4 plates, 38 cells. E, nNOS inhibition by 100 M 7NI significantly increased phospho- p38 levels at 0 min and at 5 min 10 M NMDA conditions compared with vehicle. p 0.0001 by ANOVA, p 0.05 at 0 min, and p 0.01 at 5 min, 10 M NMDA, by post-hoc Student’s t test with Bonferroni correction. However, 7NI did not alter the phosphorylation or dephosphorylation profile of NMDAR-mediated p38 activa- tion as 10 M NMDA still led to p38 acti- vation and 100 M NMDA led to activa- tion followed by deactivation (compiled from four experiments). F, using antibod- ies specific to phospho-ERK1/2, 10 M KN62 attenuated phospho-ERK1/2 im- munoreactivity with 5 min of 10 M NMDA treatment (inset), but did not af- fect phospho-p38 levels or total p38 with any treatment condition (compiled from four experiments).

Article Snippet: All inhibitors were acquired from Sigma-Aldrich, unless otherwise indicated, and included MK801 (1 M), Ro25-6981 (10 M), zinc (500 nM), conantokin G (ConG; 6 M; generous gift from Drs. Francis Castellino and Mary Prorok, Notre Dame), FK506 (1 M; generous gift from Dr. Ted Dawson, Johns Hopkins University), KN62 (10 M), 7-nitroindazole (7NI; 100 M), LY294002 (100 M; Tocris Cookson, Balwin, MO), ifenprodil (1 M), dantrolene (10 M), wortmannin (10 nM and 100 nM), and SB203580 (1 M).

Techniques: Inhibition, Activation Assay, Control, Two Tailed Test, Imaging, Phospho-proteomics, De-Phosphorylation Assay