Journal: EMBO Reports
Article Title: Linear ubiquitination by LUBEL has a role in Drosophila heat stress response
Figure Lengend Snippet: LUBEL catalyzes the synthesis of linear Ub chains In vitro ubiquitination assays of LUBEL‐RBR‐C WT or catalytically dead C2704A in combination with Ube1 and two different Drosophila E2s, UbcD10, or Effete/UbcD1. Reactions were terminated at indicated times, and synthesized Ub chains were detected by immunoblotting using anti‐linear Ub antibody. Total protein loading was visualized by Ponceau S staining. *: nonspecific band. In vitro deubiquitination of Ub chains synthesized by HOIP‐ and LUBEL‐RBR‐C. Ub chains produced by RBR‐C (human or Drosophila ) with UbcH7, UbcD10 or Effete were incubated with a linear linkage‐specific DUB, OTULIN (WT or catalytically dead C129A (C/A) mutant), or a Lys‐linkage‐specific DUB, vOTU. The DUB‐treated samples were subjected to Coomassie staining or immunoblotting using anti‐Ub antibody. *: nonspecific band. Thioester formation assay using Atto647‐labeled Ub. Ubiquitination assay using LUBEL‐RBR‐C WT (left panel) or C2704A (right panel): lanes 1/2 Atto‐Ub + E1, lanes 3/4 + ATP, lanes 5/6 + E2, lanes 7/8 + E3, and lanes 9/10 + Ub‐His 6 . In the presence of N‐terminally tagged Atto‐Ub and C‐terminally tagged Ub‐His 6 , only Ub 2 product can be synthesized and longer chain formation is restricted. Samples are run without or with DTT in odd or even numbered lanes, respectively. The gels monitor the Atto‐labeled Ub. The linear Ub chain formation activity of LUBEL‐LDD mutants. Recombinant proteins of RBR‐C WT, C2704A mutant, and LDD mutants (R2754A and D2755A) were assessed for their activity by in vitro ubiquitination assay. The samples were resolved on a gel and stained with Coomassie or immunoblotted using anti‐linear Ub antibody. Linear Ub chain formation by full‐length LUBEL transient expression in insect cells. Full‐length Myc‐LUBEL was transiently expressed in Drosophila Schneider 2 (S2) cells, and Myc‐HOIP alone or with HA‐HOIL‐1L in HEK293T cells. Total cell lysates (TCL) of control and transfected samples were incubated with immobilized GST‐Linear‐Tandem Ub binding entity (Linear‐TUBE) containing three tandem repeats of ABIN‐1‐UBAN. Pulldown samples were blotted with anti‐linear Ub antibody, while TCL were blotted with anti‐Myc antibody for exogenous LUBEL and HOIP, anti‐HA antibody for HOIL‐1L, and anti‐tubulin antibody for loading. Input of GST proteins was analyzed by Ponceau S staining. *: nonspecific band.
Article Snippet: Antibodies and reagents Antibodies used in this study are as follows: anti‐Myc antibody (clone 9E10; Covance, Princeton, NJ), anti‐Flag antibody (clone M2; Sigma, St. Louis, MO), anti‐HA antibody (HA.11 clone 16B12, Covance, Princeton, NJ), anti‐Alpha‐tubulin antibody (ab15246, Abcam, Cambridge, UK), anti‐Linear Ub antibody (LUB9; Life Sensors, Malvern, PA), anti‐Lys 63‐Ub antibody (Apu3; Merck Millipore, Darmstadt, Germany), anti‐Lys 48‐Ub antibody (Apu2; Merck Millipore), and anti‐Ub antibody (clone P4D1; Santa Cruz Biotechnology, Santa Cruz, CA).
Techniques: In Vitro, Synthesized, Staining, Produced, Incubation, Mutagenesis, Tube Formation Assay, Labeling, Ubiquitin Assay, Activity Assay, Recombinant, Expressing, Transfection, Binding Assay