Journal: PLoS ONE
Article Title: Genetic and Proteomic Evidence for Roles of Drosophila SUMO in Cell Cycle Control, Ras Signaling, and Early Pattern Formation
Figure Lengend Snippet: A fly SUMO-ome: characterization and validation. A) Scheme for identifying Drosophila embryonic SUMO conjugates. SUMO conjugates were isolated by tandem affinity purification from transgenic fly embryos expressing (His) 6 -FLAG-SUMO. The initial purification step (Ni-NTA chromatography) was performed under denaturing conditions. To maximize the chance of detecting low abundance proteins in the complex protein mixture, the affinity-purified proteins were separated by SDS-PAGE, and the lane was cut into 20 evenly divided gel slices. Tryptic peptides extracted from each gel slice were analyzed by LC-MS/MS. B) A bacterial sumoylation assay. The Q SUMO vector, which encodes the mature form of SUMO (SUMO GG ) along with SAE1, SAE2, and Ubc9 expressed from separate T7/lac promoters, was used in combination with a vector expressing a GST-tagged candidate substrate. As a negative control, Q ΔGG , which expresses a conjugation defective form of SUMO (SUMO ΔGG ), was used in place of Q SUMO . C) Bacterial sumoylation assays were used to validate proteins identified in the proteomic screen as sumoylation substrates. GST-tagged candidate SUMO conjugates were expressed in BL21 cells co-transformed with Q SUMO or Q ΔGG vectors, purified using glutathione beads, and immunoblotted using antibodies against GST, SUMO, or poly-His (to detect 6xHis-tagged SUMO). GST by itself was not sumoylated in this assay. Black arrows point to the bands representing sumoylated proteins, and open arrow points to a non-specific reacting band. D) The eIF4E protein was purified from Drosophila S2 cells stably expressing FLAG-(His) 6 - tagged eIF4E using Ni-NTA beads under denaturing conditions. The resulting proteins were probed with anti-FLAG antibody in a Western blot. The cells were treated with SUMO or control YFP dsRNA for 3 days prior to cell lysis. In the control sample, the bands representing the sumoylated species (black arrows) have intensities that are 8.1% (top) and 12.9% (bottom) of the intensity of the band representing unmodified eIF4E (∼40 kDa), whereas in the SUMO knockdown sample, they are reduced to 1.8% (top) and 3.5% (bottom). Quantitation was performed using Quantity One 4.3.0 (BioRad).
Article Snippet: The bacterial sumoylation assay A vector encoding a candidate SUMO conjugation target fused to GST was co-transformed into BL21 cells (Novagen) with either the QSUMO or the control QΔGG expression vectors.
Techniques: Isolation, Affinity Purification, Transgenic Assay, Expressing, Purification, Chromatography, SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Plasmid Preparation, Negative Control, Conjugation Assay, Transformation Assay, Stable Transfection, Western Blot, Lysis, Quantitation Assay