Antibodies used are listed in Table S8 . Yeast cell lysates were prepared as described previously (Fei et al., 2008 (link)). 2 mg protein was used for each co-immunoprecipitation condition. Antibody was immobilized on magnetic Dynabeads (Dynabeads Co-Immunoprecipitation Kit manual). After 30 min of incubation with the cell lysates containing overexpressed protein at RT, or after overnight incubation with the cell lysates containing low levels of recombinant protein at 4°C, beads were washed and eluted at 37°C for 20 min. Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes, followed by immunodetection (Fei et al., 2008 (link)).
Transiently or stably transfected mammalian cells (MEFs, mouse 3T3-L1preadipocytes, and HeLa cells) were washed three times with ice-cold PBS and lysed in immunoprecipitation (IP) lysis/wash buffer (as above) containing 1% (w/v) n-dodecyl-β-D-maltopyranoside (DDM; Sigma) by forcing the cells 30 times through a 0.8-mm needle. Immunoprecipitation and western blotting were carried out as described above.
Transiently or stably transfected mammalian cells (MEFs, mouse 3T3-L1preadipocytes, and HeLa cells) were washed three times with ice-cold PBS and lysed in immunoprecipitation (IP) lysis/wash buffer (as above) containing 1% (w/v) n-dodecyl-β-D-maltopyranoside (DDM; Sigma) by forcing the cells 30 times through a 0.8-mm needle. Immunoprecipitation and western blotting were carried out as described above.