Polyubiquitin chains

The 293T cells were transfected with 1 μg of Snail, HA-ubiquitin and WT STAMBPL1 or D360A STAMBPL1 plasmids. Lysates were subjected to immunoprecipitation (IP) with anti-Snail and immunoblotting with anti-ubiquitin to examine ubiquitination level of Snail. For in vitro DUB assay: Deubiquitination cleavage assays were performed using recombinant GST-tagged STAMBPL1 (Ubiquigent) and polyubiquitin chains (BostonBiochem). STAMBPL1 were freshly prepared in assay buffer (40 mM Tris-Hcl, pH 7.5, 5 mM dithiothreitol (DTT), 0.005% bovine serum albumin) and activated for 10 min at 30 °C. Subsequently, 1 μg of STAMBPL1 were incubated with 200 ng of either Lys⁶³ or Lys⁴⁸ polyubiquitin (Ubi₁–Ubi₇) chains at 30 °C for the indicated time points. The reactions were stopped by the addition of SDS sample buffer (Bio-Rad) supplemented with 100 mM freshly prepared DTT). Ubiquitin cleavage was detected using immunoblotting and anti-ubiquitin antibodies (Enzo, BML-PW8810).

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). A rabbit polyclonal antibody recognizing LUBEL‐RBR was generated using recombinant LUBEL‐RBR (aa 2,514–aa 2,655) (immunoGlobe, Himmelstadt, Germany). His₆ Ube1, ubiquitin, His₆‐ubiquitin, di‐ubiquitin chains (Lys 6, Lys 11, Lys 27, Lys 29, Lys 33, Lys 48, Lys 63‐linked and linear chains), poly‐ubiquitin chains (2–7, Lys 48 and Lys 63‐linked chains, and tetra linear chains) were purchased from Boston Biochem (Cambridge, MA).

HEK293T cells were cotransfected with vectors expressing FLAG-tagged HsCYLD, CeCYLD or CeCYLDC774S, using the calcium phosphate transfection method. The cells were harvested approximately 18 h post transfection and lysed in 1% NP40 lysis buffer (50mM Tris–HCL pH 7.4, 150mM NaCl, 1mM EDTA, 10% Glycerol, 1% NP-40, 1mM DTT, 1mM PMSF and 1× protease inhibitor cocktail (Roche, 1697498)). FLAG-tagged proteins were immunoprecipitated with the M2 anti-FLAG mouse monoclonal antibody (Sigma, F3165). The immunoprecipitated products were then divided equally and incubated in vitro with Poly-ubiquitin chains (Ub_3-7, K63-linked) or Recombinant Human Tetra Ubiquitin/Ub4 WT Chains (M1 linked) (Boston Biochem, UC-320 and UC-710b respectively). The hydrolysis products were separated from the enzymes and boiled in 1xSDS loading buffer.