Fig. 1.

LUBEL is required for signal-dependent M1-Ub chain formation in flies. a Adult wild-type Canton^S and lubel^Mi mutant flies were infected by septic injury with the Gram-negative bacteria Ecc15. M1-Ub chains were isolated at denaturing conditions from fly lysates with GST-NEMO-UBAN. Ubiquitin chains from lysates and pulldown samples were analysed by Western blotting with α-M1 and pan-ubiquitin antibodies and equal loading was controlled with α-Actin antibody, n = 9. b Adult wild-type Canton^S and lubel^Mi flies were starved for 2 h before infection by feeding with the Gram-negative bacteria Ecc15. Canton^S flies fed with LB were used as controls. M1-Ub chains were isolated from fly lysates with GST-NEMO-UBAN at denaturing conditions. Ubiquitin chains from lysates and pulldown samples were analysed by Western blotting with α-M1 and pan-ubiquitin antibodies and equal loading was controlled by immunoblotting with α-Actin antibodies, n = 3. c Adult wild-type Canton^S and lubel^Mi mutant flies were infected by septic injury with the Gram-negative bacteria Ecc15. M1-Ub chains were isolated from fly lysates with GST-NEMO-UBAN. The GST-NEMO-UBAN-isolated samples were subjected to ubiquitin chain restriction (UbiCRest) with recombinant OTULIN and vOTU and M1-Ub chains were analysed by Western blotting with an α-M1 antibody, n = 3