Nupage sds page sample loading buffer

Cells were lysed in 25 mM Tris.HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Igepal CA‐630 (Sigma‐Aldrich) and 5% glycerol, protease inhibitors (Cell Signalling) and phosphatase inhibitor cocktail 3 (Sigma). Protein lysates were cleared by centrifugation at 16,000 g for 10 min. Alternatively, cells were directly lysed in NuPAGE SDS–PAGE sample loading buffer (ThermoFisher). 50 mM of the reducing agent DTT was added to the lysates, except for non‐reducing gels. Protein lysates were run on Bis‐Tris SDS–PAGE gels (NuPAGE, ThermoFisher). Primary antibodies used for Western blotting are listed in Appendix Table S3. Anti‐rabbit, anti‐rat or anti‐mouse secondary antibodies coupled to HRP were from GE Healthcare.

Cells were lysed in RIPA buffer (50 mM Tris.HCl, pH7.4; 150 mM NaCl; 1% NP-40 (Sigma-Aldrich), 0.5% Deoxycholate, 0.1% SDS and Complete protease inhibitor (Roche)) at 4°C for 10 minutes. Protein lysates were then cleared by centrifugation at 13000 rpm for 10 minutes. Samples were mixed with NuPAGE SDS-PAGE sample loading buffer (ThermoFisher) containing 10% 2-mercaptoethanol. A primary antibody against ADAR1 was purchased from Santa Cruz (sc-73408). The antibody recognizing ISG15 was a gift from Klaus-Peter Knobeloch. HRP-coupled secondary antibodies were from GE Healthcare.