Journal: Biochimica et Biophysica Acta. Molecular Cell Research

Article Title: A microarray of ubiquitylated proteins for profiling deubiquitylase activity reveals the critical roles of both chain and substrate ☆

doi: 10.1016/j.bbamcr.2012.05.006

Figure Lengend Snippet: DUBs examined for behavior against ubiquitylated substrates were active enzymes. A. The fourteen DUBs examined in Fig. 2 all showed activity (signal versus time over 1 h) by CHOP reporter assay (LifeSensors, Inc.), except AMSH. B. AMSH was examined for activity (versus time over 1 h) against different substrates, di-ubiquitin substrates labeled with an internally quenched fluorophore pair. As expected, activity was specific to K63-linkage of the diubiquitin. C. E3Lite assay data (endpoint RFU signal) for triplicate wells either ubiquitylated with E3 mix (Praja1, Carp2, Murf1) and then mock treated with DUB (“No DUB”); mock ubiquitylated using reaction mixture lacking an E3 and then mock treated with DUB (“Not UB'd”); or ubiquitylated with E3 mix and then treated with DUB enzyme (as listed).

Article Snippet: Since this DUB is known to prefer K63 linked chains, we incubated it with a K63 linked internally quenched fluorescence based di-ubiquitin substrate (LifeSensors, Inc.).

Techniques: Activity Assay, Reporter Assay, Ubiquitin Proteomics, Labeling

Journal: Science signaling

Article Title: Traf6 and A20 Regulate Lysine 63-Linked Ubiquitination of Beclin 1 Controlling TLR4-Induced Autophagy

doi: 10.1126/scisignal.2000751

Figure Lengend Snippet: TLR4 engagement, MyD88, Trif, and Traf6 promote the ubiquitination of Beclin 1. (A) Ubiquitination of Beclin 1. LPS was used to stimulate Raw 267.4 cells for various durations. Shown is a Beclin 1 immunoprecipitation immunoblotted for ubiquitin (Ub, left panel), Beclin 1 (middle panel), and K63-linked ubiquitin (right panel). After each immunoblotting the membrane was stripped and the efficacy of the stripping verified. The major non-specific band (*) results from the secondary antibody recognizing the immunoprecipitating antibody. Lysates were immunoblotted as indicated. LPS stimulation reduced IκB levels in the cell lysates as expected. (B) Overexpression of MyD88 or Trif enhances Beclin 1 ubiquitination. FLAG immunoprecipitates from cells expressing MyD88 or Trif with FLAG-Beclin 1 were immunoblotted for ubiquitin, stripped and re-blotted for Beclin 1. Lysates immunoblotted as indicated. (C) Bcl-2 attenuates while Traf6 overexpression enhances Beclin 1 ubiquitination. FLAG immunoprecipitates from Raw 264.7 cells expressing Bcl-2 or Traf6 with FLAG-Beclin 1 were immunoblotted for ubiquitin, stripped, and re-blotted for Beclin 1. Lysates were immunoblotted as indicated. (D) Reduced Traf6 expression impairs Beclin 1 ubiquitination. Beclin 1 immunoprecipitates from Raw 264.7 cells expressing control or shRNAs targeted at Traf6 were immunoblotted for ubiquitin, stripped, and re-blotted for Beclin 1. The lysates were immunoblotted as indicated.

Article Snippet: After washing with lysis buffer 8 times, K63- or K48-linked ubiquitin chain peptides (BIOMOL) 0.5 μg for each sample were added to the samples, and then the samples were incubated by rotating at 4 °C for 1 hour.

Techniques: Immunoprecipitation, Western Blot, Stripping Membranes, Over Expression, Expressing

Journal: Science signaling

Article Title: Traf6 and A20 Regulate Lysine 63-Linked Ubiquitination of Beclin 1 Controlling TLR4-Induced Autophagy

doi: 10.1126/scisignal.2000751

Figure Lengend Snippet: Traf6 binds Beclin 1 and triggers in vitro ubiquitination of Beclin 1. Traf6 binding sites identified in Beclin 1. FLAG-Traf6 was expressed along with various GST Beclin 1 fusion proteins. GST pull-downs were immunoblotted for GST, GST-Beclin 1, various GST-Beclin 1 mutants, and FLAG-Traf6. Lysates were blotted for Traf6. (B) Traf6 binding mutants of Beclin 1 undergo less ubiquitination. Following stringent washing the indicated GST pull-downs were immunoblotted for K63-linked ubiquitin. The blot was stripped and re-blotted for GST to detect GST-Beclin 1 and the various GST-Beclin 1 mutant proteins. Lysates were blotted for FLAG. (C) FLAG-Traf6 catalyzes FLAG-Beclin 1 ubiquitination in vitro. Purified proteins along with the indicated reaction components were combined and incubated. After which Beclin 1 immunoprecipitates from each reaction were analyzed by immunoblotting them for HA-ubiquitin (HA-Ub). Saved portions of each of the reactions were immunoblotted for FLAG to detect Beclin 1 and Traf6.

Article Snippet: After washing with lysis buffer 8 times, K63- or K48-linked ubiquitin chain peptides (BIOMOL) 0.5 μg for each sample were added to the samples, and then the samples were incubated by rotating at 4 °C for 1 hour.

Techniques: In Vitro, Binding Assay, Mutagenesis, Purification, Incubation, Western Blot

Journal: Science signaling

Article Title: Traf6 and A20 Regulate Lysine 63-Linked Ubiquitination of Beclin 1 Controlling TLR4-Induced Autophagy

doi: 10.1126/scisignal.2000751

Figure Lengend Snippet: A20 regulates the ubiquitination of Beclin 1. (A) Reduced A20 expression promotes LPS-induced ubiquitination of Beclin 1. Beclin 1 immunoprecipitates from Raw 264.7 cells expressing indicated shRNAs were blotted for ubiquitin (Ub), stripped and re-blotted for Beclin 1. The major non-specific bands (*) are indicated. (B) A20 and its E3 ligase mutant, but not the deubiquitination dead form attenuate LPS-induced ubiquitination of Beclin 1. Cell lysates and Beclin 1 immunoprecipitates from cells expressing A20 or various mutants were immunoblotted for as indicated. (C) Interaction between Beclin 1 and A20. Cell lysates and Beclin 1 immunoprecipitates from Raw 264.7 cells stimulated with LPS (1 hr) or not were immunoblotted as indicated. (D) A20 deubiquitinates Beclin 1. Purified FLAG-Beclin 1 was incubated with recombinant A20 (1–371) and the reactions immunoblotted as indicated.

Article Snippet: After washing with lysis buffer 8 times, K63- or K48-linked ubiquitin chain peptides (BIOMOL) 0.5 μg for each sample were added to the samples, and then the samples were incubated by rotating at 4 °C for 1 hour.

Techniques: Expressing, Mutagenesis, Purification, Incubation, Recombinant

Journal: Science signaling

Article Title: Traf6 and A20 Regulate Lysine 63-Linked Ubiquitination of Beclin 1 Controlling TLR4-Induced Autophagy

doi: 10.1126/scisignal.2000751

Figure Lengend Snippet: Beclin 1 binds ubiquitin. (A) GST-Beclin 1 preferentially binds K63-linked ubiquitin. GST and GST-Beclin 1 pull-downs were incubated with K63- or K48-linked peptides, washed, and analyzed by immunoblotting for Ub. The blot was stripped and re-probed for GST to identify GST or GST-Beclin 1. (B). Endogenous Beclin 1 preferentially binds K63-linked ubiquitin. Beclin 1 immunoprecipitates from Raw 264.7 cells were similarly evaluated for K63- and K48-linked Ub peptide binding. The initial blot was stripped and re-immunoblotted for Beclin 1.

Article Snippet: After washing with lysis buffer 8 times, K63- or K48-linked ubiquitin chain peptides (BIOMOL) 0.5 μg for each sample were added to the samples, and then the samples were incubated by rotating at 4 °C for 1 hour.

Techniques: Incubation, Western Blot, Binding Assay

Journal: Science signaling

Article Title: Traf6 and A20 Regulate Lysine 63-Linked Ubiquitination of Beclin 1 Controlling TLR4-Induced Autophagy

doi: 10.1126/scisignal.2000751

Figure Lengend Snippet: Mutation of Lysine 117 in Beclin 1 reduces Beclin 1 ubiquitination. (A) Amino acid alignment of a portion of the BH3 domains of different species. (B) Contrast between LPS-induced ubiquitination of Beclin 1 and its K117R mutant. FLAG immunoprecipitates of Raw 264.7 cells expressing FLAG-Beclin 1 or FLAG-K117R Beclin 1 were immunoblotted for K63 linked ubiquitin (K63 Ub), stripped, and re-blotted for Beclin 1. The major non-specific bands (*) are indicated. The heavy chain of the FLAG antibody was detected by the secondary antibody in the left panel and is labeled Ig band. The density of each lane over the indicated area is shown. (C) Beclin 1 K117R is poorly ubiquitinated in vitro. Purified FLAG-Beclin 1 or FLAG-Beclin 1 K117R was incubated with eluted FLAG-Traf6 along with reaction components. The reactions were immunoblotted for HA-Ub. Similar amounts of Traf6 and the FLAG-tagged proteins were in the reaction. (D) Beclin 1 ubiquitination enhances Beclin 1 oligomerization. GST-pull downs from Raw 264.7 cells expressing GST-Beclin 1 and FLAG-Beclin 1 or GST-Beclin 1 K117R and FLAG-Beclin 1 K117R stimulated with LPS, or not, were immunoblotted for FLAG. (E) Beclin 1, but not its K117R mutant, enhances the activity of PI3KC3 in Raw 264.7 cells. GFP-2xFYVE dots were imaged. Beclin 1 or Beclin 1 K117R was expressed in Raw 264.7 cells along with GFP-2xFYVE. The cells were imaged and then treated with 5mM 3-MA or DMSO for 2 h. The cells were then re-imaged. Representative cells are shown (top panel). The average number of dots in 100 cells from the indicated each sample was determined. Data displayed as mean +/− SD of the number of dots/cell (bottom panel). Similar results in 3 experiments performed.

Article Snippet: After washing with lysis buffer 8 times, K63- or K48-linked ubiquitin chain peptides (BIOMOL) 0.5 μg for each sample were added to the samples, and then the samples were incubated by rotating at 4 °C for 1 hour.

Techniques: Mutagenesis, Expressing, Labeling, In Vitro, Purification, Incubation, Activity Assay