To detect SUMOylated FOG-2 and its derivatives, Western blot analyses were carried out in the existence of the deSUMOylation inhibitor N-ethylmaleimide (NEM) at a ultimate concentration of 25 mM essentially as beforehand described [19]. For transfected COS-seven cells, nuclear extracts [19] or whole cell lysates ended up analyzed. Membranes had been probed with the following primary antibodies (Ab): rabbit anti-FOG-two Ab (sc-10755 Santa Cruz 1:200), rabbit anti-GATA-4 (sc-9053 Santa Cruz one:five hundred) mouse anti-b-actin (clone AC-fifteen, Sigma, 1:5000), rabbit anti-GFP (Ab290, Abcam one:5000) and mouse anti-SUMO-one (one:300). The Baicalinsecondary antibodies used had been anti-rabbit-HRP Ab (sc-2054 Santa Cruz one:5000 dilution) and anti-mouse HRP Ab (P0260 of five% CO2, 95% air. Neonatal rat cardiomyocytes had been obtained from Lonza and cultured adhering to the manufacturer’s instructions (Lonza, Waverly, VIC, Australia).
Mouse myoblast C2C12 cells, African eco-friendly monkey kidney fibroblasts (COS-seven cells) and HeLa cells were used for transfections. Cells ended up acquired from ATCC (Rockville, MD). Cells have been cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen) supplemented with ten% dialyzed fetal bovine serum (Invitrogen) and preserved at 37uC in a humidified ambiance predicted residues are component of non-consensus sequences (Table 1). The putative SUMOylated lysines in the consensus sequences were mutated to arginine and vectors encoding these constructs were transfected into COS-seven cells in the existence or absence of HA-SUMO-one. Fig. 2B displays that equally wild-kind and the mutants K324R, K471R or K915R were SUMOylated by HA-SUMO-one, suggesting that there might be a lot more than one particular acceptor website in FOG2. It is evident in Fig. 1A that FOG-two is being modified by much more than a single SUMO-1 moiety (Fig. 1A, arrowheads). However, the large molecular mass of FOG-2 precluded unambiguous separation of the SUMOylated species as SUMO-1 will increase the obvious molecular mass of modified proteins by only around 20 kDa. For this purpose, COS-seven cells had been co-transfected with expression vectors for FOG-two and a GFP-SUMO-one fusion that boosts the dimensions of the SUMO moiety to roughly fifty kDa. At least 3 slower migrating species ended up observed (Fig. 2C, lane 2, arrowheads) indicating that far more than two lysine residues in FOG-2 could be qualified by SUMO-1. A amount of solitary and combination mutants were created and then expressed in COS7 cells and analyzed by Western blot. Fig. 2C, lanes 3, shows a choice of these mutants. Combinations of double and triple mutants unveiled that all SUMOylation bands, apart from one particular, ended up abolished when lysine residues 324, 471 and 915 were mutated to arginine (Fig. 2C, lane 6). Mutation of numerous other residues that also experienced a higher theoretical likelihood of getting SUMOylated these kinds of as K729 and K1049 in conjunction with residues 324, 471 and 915 did not avert the visual appeal of a one SUMOylation band (information not demonstrated). To outline the region of the very last SUMOylation internet site of FOG-2, a collection of deletion mutants was designed and then12084461 subjected to SUMOylation in COS-7 cells (Fig. 3A). Fragments 509151 and 729151 were SUMOylated at seemingly a single website (Fig. 3A, FOG-2(509151), lane 3 and FOG-2(729151), lane 2). One stage mutations in the 729151 fragment did not fully abrogate SUMO modification (Fig. 3A, FOG-2(7291151)KR mutants, lanes three). Additional mutations in the fragment made up of amino acids 881092 of FOG-two discovered K955 as a SUMO-acceptor residue, and mutation of this amino acid, in combination with K915, resulted in the comprehensive elimination of SUMOylation of this FOG-2 fragment (Fig. 3A, FOG-2(881092), lane 4). The validity of this outcome was then confirmed in the context of the complete-duration protein. Fig. 3B shows that the quadruple mutant (from now on termed FOG-2-4KR) is not modified by SUMO-1 (Fig. 3B, lane 9, higher panel). As a result K324, K471, K915 and K955 are the only SUMO acceptor websites in mouse FOG-2. The mapping of the FOG-2 SUMOylation sites was verified by immunoprecipitation of SUMOylated FOG-two wt, K324/471/ 915R and 4KR. Fig. 4A exhibits that at least two SUMOylated bands are pulled down by the anti-GFP antibody when FOG-two wt is SUMOylated by GFP-SUMO-one (Fig. 4A, lane two, upper panel).
