17608 deposited by ted dawson Search Results


ted dawson  (Addgene inc)
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Addgene inc ted dawson
Ted Dawson, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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ha tagged ubiquitin  (Addgene inc)
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Addgene inc ha tagged ubiquitin
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 <t>Ubiquitin.</t> 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.
Ha Tagged Ubiquitin, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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plasmids 17608 17605  (Addgene inc)
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Addgene inc plasmids 17608 17605
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 <t>Ubiquitin.</t> 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.
Plasmids 17608 17605, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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.

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