Hoil 1

Cells were washed with ice-cold PBS, lysed with TNT buffer and incubated for 30 min on ice.
Extracts were cleared by centrifugation at >10,000 g. Protein concentration was determined using the Micro BCA protein kit. Samples were precleared with Protein G Sepharose (Merck) for 30 min, and subsequently incubated with 1 g of antibody and Protein G Sepharose for a duration of 2h. Flag pull down was performed incubating cleared cell lysates with Anti-flag M2 affinity gel (Merck) during 2h. SHARPIN (A303-559A, Bethyl), HOIL-1 ((sc-393754, Santa Cruz Biotechnology) and linear ubiquitin, kindly provided by VM Dixit (Genentech), antibodies were used for IP. ERK1/2 kinase assay was performed from beads immunoprecipitated for HOIL-1. Beads were washed in kinase buffer (5 mM MOPS pH 7.2, 5 mM MgCl 2 , 1 mM EGTA, 0.4 mM EDTA, 0.05 mM DTT). Beads were subsequently incubated with kinase buffer, 2mM ATP, 350 ng/mL active untagged ERK1 (Merck), 350 ng/mL active ERK2-GST tagged (Merck) or 175 ng/mL ERK1 plus 175 ng/mL ERK2-GST for 30 min at 30°C.