Immunoprecipitation

NIH3T3 cells were lysed in a buffer containing 50 mM Tris-Hcl pH 7.5–8, 150 mM NaCl, 1%Triton X-100 and protease and phosphatase inhibitors and homogenized for 30 min in a rotor wheel. 3 mg of lysates were immunoprecipitated with 5 μg of monoclonal HA-tag antibody (Sigma) overnight at 4°C. Immunocomplexes were collected using PureProteome™ Protein A Magnetic Beads (MERCK) and eluted by boiling for 5 min in SDS-loading buffer. Western blotting, as described above, was used to visualize pTUNAR (HA-tag antibody) and SERCA2 (SERCA2 ATPase antibody). Antibodies and dilutions are listed in Supplementary Table S2.