Fig. S1.

Bam is an ubiquitin binding protein. (A) MS-identified Bam-associating proteins in transfected S2 cells (n = 3). In this assay, S2 cells were transfected with Flag-GFP or Flag-Bam plasmids. After 48 h, cells were harvested and lysed, followed by immunoprecipitation with anti-Flag M2 beads. The immunoprecipitants were eluted and digested by using Trypsin (Promega), and then were subjected to LC-MS/MS assays. Resulting MS/MS data were processed by using Thermo Proteome discovery (version 1.4.1.14), and tandem mass spectra were searched against UniProt-Drosophila database. Unique peptides that were detected only in Flag-Bam immunoprecipitants, or displayed at least 2 folds higher abundance than the Flag-GFP control groups, were selected. From all these peptides, only the ones that emerged in all of the three replicates were considered as Bam associated proteins. (B) HA-Ubiquitin proteins were mixed and incubated with different truncated Bam proteins as indicated. The incubated products were then subjected to immunoprecipitation assays by using GST beads. Western blot assays were performed to detect the indicated proteins.