Identifying Protein Interactions via IP-MS

HEK293 cells were transfected with PGK::FLAG-GIG or PGK::FLAG-BTB constructs. 28 hours later, cells were lysed and immunoprecipitation was performed as previously described [29 (link)]. Targets were eluted by incubation with 3X flag peptide (Sigma). Samples were analyzed by Applied Biomics, Inc (Hayward, CA) via NanoLC-MS/MS after tryptic digestion, and only high confidence hits (CI > 95%) were considered for analysis. CRAPome analysis was performed as previously described [30 (link)]. DAVID analysis was performed using the published online tool [17 (link)]. For follow-up immunoprecipitation experiments, the same strategy was used to isolate flag-GIG. Endogenous GIG was immunoprecipitated using rabbit α GIG (NBP1-49924, Novus Biologicals). Endogenous VIM was immunoprecipitated using mouse α VIM (BD 550513 or AMF-17b). The mouse peripherin construct was a generous gift from Jean-Pierre Julienne (Université Laval).