Protease inhibitor cocktail set 5

HeLa cells transfected with vectors expressing IDS and HA tagged with ubiquitin were lysed in cell lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 5 mM EDTA and Protease inhibitor cocktail Set V (Wako)] for 20 min. Supernatants were incubated with anti-IDS (goat IgG, 1:2000; R & D Systems, RRID: AB_2123559) antibodies and Protein G Agarose Beads (Invitrogen) at 4 °C for 4 h. The beads were rinsed five times with a wash buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Triton X-100]. Immunoprecipitates were boiled with SDS-PAGE sample buffer, followed by western blotting using the antibodies: anti-IDS (mouse IgG, 1:2000; R & D Systems, RRID: AB_2233551), anti-ubiquitin (mouse IgG, 1:1000; Cell Signaling Technology, RRID: AB_10998070) and anti-HA (rabbit IgG, 1:1000; Cell Signaling Technology, RRID: AB_1549585).

Cell pellets from T-REx Mock and T-REx AspALP cells were lysed in Cell Lysis Buffer M (FUJIFILM Wako Pure Chemical Corporation) supplemented with Protease Inhibitor Cocktail Set V (EDTA free) (FUJIFILM Wako Pure Chemical Corporation) at 1 × 10⁷ cells/mL. Dynabeads protein A (Thermo Fisher Scientific) was mixed with anti-CaM antibodies (Abcam) and formed the magnetic bead-antibody complex. The complex was mixed with 200 μL of cell lysates in the presence or absence of 10 mM EGTA with gentle rotation. The magnetic bead-antibody-antigen complex was washed 3 times. Regarding Western blotting, the complex was supplemented with 30 μL of SDS loading buffer (Cell Signaling Technologies) and denatured at 95 °C for 5 min. In the MS analysis, the complex was lysed in 100 μL of 8 M urea.

The interaction of RNF183 and DR5: HEK293 cells stably expressing FLAG-tagged RNF183 were lysed in lysis buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 100 μM MG132 (FUJIFILM Wako Pure Chemical), Protease inhibitor cocktail Set V (EDTA free; FUJIFILM Wako Pure Chemical) on ice for 20 min. Supernatants were incubated with an anti-FLAG antibody at 4 °C for 1 h, and followed by incubation with Protein G Agarose Beads (Thermo Fisher Scientific) for 1 h; subsequently, the beads were rinsed three times with a wash buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 0.1% Triton X-100]. Immunoprecipitates were boiled with Laemmli sodium dodecyl sulphate polyacrylamide gel electrophoresis sample buffer and analysed by western blotting.
The ubiquitination of DR5:HEK293 cells stably expressing V5-tagged RNF183 were lysed in lysis buffer containing 20 µM MG132, 1 mM N-ethylmaleimide (FUJIFILM Wako Pure Chemical), 5 mM EDTA on ice for 20 min, and then quenched with 0.1% cysteine. Supernatants were incubated with anti-DR5 antibody at 4 °C for 1 h, followed by incubation with Protein G Agarose Beads for 1 h; the beads were rinsed with the same wash buffer.