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: Nature Communications

Article Title: The ubiquitin ligase RNF5 determines acute myeloid leukemia growth and susceptibility to histone deacetylase inhibitors

doi: 10.1038/s41467-021-25664-7

Figure Lengend Snippet: a Schematic showing full-length and mutants forms of RNF5. b Immunoprecipitation (IP) and Western blot (WB) analysis of HEK293T cells transfected with Flag-tagged forms of full-length RNF5 (WT), the catalytically inactive RING domain mutant (RM), or the C-terminal transmembrane domain deletion mutant (∆CT). Cells were treated with MG132 (10 µm, 4 h) before lysis. c IP and WB of HEK293T cells co-expressing Myc-RBBP4 and Flag-tagged RNF5-WT treated with MG132 (10 µm, 4 h) before lysis. d IP and WB of ectopically expressed doxycycline-inducible Flag-tagged RNF5 and endogenous RBBP4 in MOLM-13 cells. Cells were incubated for 2 days with or without doxycycline (1 µg/ml) and with MG132 (10 µm, 4 h) before lysis. e WB of anti-Myc IP from lysates of HEK293T cells co-expressing Myc-RBBP4, hemagglutinin-tagged ubiquitin (HA-Ub), and indicated Flag-tagged RNF5 constructs. Cells were treated with MG132 (10 µm, 4 h) before lysis. f WB of indicated proteins in MOLM-13 cells expressing empty vector (pLKO) or indicated shRNF5 construct. g WB analysis of indicated proteins in MOLM-13 cells expressing empty vector or doxycycline-inducible Flag-tagged RNF5. h WB of anti-Myc IP and lysates of HEK293T cells co-expressing Myc-RBBP4, Flag-tagged RNF5, and different HA-tagged ubiquitin mutants (K29, K11, K6, K27, and K33). MG132 (10 µm, 4 h) was added before lysis. i IP and WB for the interaction of RBBP4 with HDAC1, HDAC2, or EZH2 in MOLM-13 cells expressing indicated constructs. j IP and WB for RBBP4 interaction with HDAC1, HDAC2, or EZH2 in MOLM-13 cells expressing indicated constructs. MG132 (10 µm, 4 h) was added before lysis. k ChIP and qPCR reveal the enrichment of RBBP4 (normalized to input) at indicated gene promoters in MOLM-13 cells expressing indicated constructs. l – n ChIP and qPCR reveal the enrichment of H3K9ac, H3K27ac, or H3K27me3 (normalized to input) at indicated gene promoters in MOLM-13 cells expressing indicated constructs. Data in k and l are presented as mean ± SEM of n = 4 ( ANXA1 ) or n = 3 ( NCF1 and CDKN1A ) independent experiment. Data in m and n are the mean of n = 2 independent experiments. The P values were determined using paired two-tailed t -test ( k and l ). Source data are provided as a Source Data file.

Article Snippet: Expression vectors encoding Myc-RBBP4 (#20715), HA-Ubiquitin (#18712), and HA-ubiquitin mutants (including K6 (#22900), K11 (#22901), K27 (#22902), K29 (#22903), and K33 (#17607)) were obtained from Addgene.

Techniques: Immunoprecipitation, Western Blot, Transfection, Mutagenesis, Lysis, Expressing, Incubation, Ubiquitin Proteomics, Construct, Plasmid Preparation, Two Tailed Test