Figure 2. Co-immunoprecipitation assay to validate interaction of ZFYVE27 in mammalian cells.

(A) Schematic representation of full-length and deletion constructs of ZFYVE27 for mammalian expression studies. (B) The full-length ZFYVE27 fused with c-Myc epitope tag was used for validation of its interaction with E2 tagged ZFYVE27, N-terminus (ΔC), C-terminus (ΔN) as well as with ZFYVE27 lacking the third hydrophobic region (ΔHR3) in NIH-3T3 cells. The cells were transiently transfected with respective constructs and subsequently co-immunoprecipitation was performed. The cell lysates were subjected to immunoprecipitation with E2 tag antibody and the resulting immunoprecipitants were analyzed in the immunoblot with c-Myc tag antibody (Co-IP: E2; WB: c-Myc). A portion of the cell lysates (input) was also subjected to immunoblot with either c-Myc (WB: c-Myc) or E2 (WB: E2) tag antibodies to verify the protein expression of the indicated constructs. For mock experiments, the cell lysates were precipitated with non-specific IgG and subsequently analyzed by immunoblotting (as described above).