The photos of the colonies developed in soft agar were taken at day 21. GE Healthcare Life Sciences. Puromycin (#P8833) was from Sigma. Anti-RPTP (#7C091) is kept in our lab [28]. Plasmids The human Src CDS was cloned from pCMV-Tag2B-Src plasmid [15], digested with EcoR I and Not I and then subcloned into vector pEF5HA, CD513B, pGEX-4 T-1 and mutant Src K318R was generated using PCR-directed mutagenesis and sequenced. The shRNA sequence targeting Src 3UTR (shSrc) was from Sigma-Aldrich Mission shRNA online: 5-CATCCTCAGGAACCAACAATT-3. The shRNA was cloned into pLKO.1 vector. The pE1E2S1 plasmid was a kind gift from Dr. Jiemin Wong in East China Normal University. Cell Culture HEK293T, HEK293FT, NIH/3 T3 and DU145 cell lines were obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium(DMEM) containing 10% fetal calf serum (Hyclone) at 37 C in 5% CO2 humidified incubator. Cell transfection was performed using Lipofectamine 2000 (Invitrogen). SUMOylation Assays Src SUMOylation was analyzed in HEK293T by the method of SUMOylation assay using Ni2+-NTA agarose beads as previously described [29]. Src SUMOylation analysis was also performed by the method of BL21-based SUMOylation assay with the plasmid pE1E2S1 as described [30]. Soft Agar Colony Assay The method was performed in six-well plates with a base of 2 ml of DMEM medium containing 5% FBS with 0.6% Bacto agar (Amresco). Stable Sirt4 NIH/3 T3 or DU145 cells were seeded in 2 ml of medium containing 5% FBS with 0.35% agar at 2 or 4 103 cells per well and layered onto the base. The photos of the colonies developed in soft agar were taken at day 21. Three independent experiments were performed in triplicate. Migration Assay by RTCA-DP The method was carried out as described previously [23]. Briefly, stable NIH/3 T3 cells were starvation pre-treated with serum-free medium for 12 hours and then 4104 cells resuspended in 100 l of serum-free medium were FRAX597 added into the pre-equilibrated upper chambers of the FRAX597 CIM-plate. The lower chamber was filled with 160 l of normal growth medium containing 10% FBS. The kinetic cell indexes of their migration were recorded every 15 min for 2 days. Mouse Xenograft Models Murine xenograft models were established as described previously [31]. Briefly, 5-weeks-old nude mice were subcutaneously injected in the back with 100 l of medium containing 2.5106 DU145 cells stably re-expressing Src WT and Src K318R. Forty-two days after injection, at the experimental endpoint, FRAX597 mice were sacrificed and the tumors were weighted and photographed. Statistical differences between groups were analyzed by the two-tailed Student’s test. and (Figure 1by a prokaryotic SUMOylation assay with pE1E2S1 [32]. pE1E2S1 is a tri-cistronic plasmid for the overexpression of SUMO-E1 enzyme (AOS1/UBA2), E2 enzyme (UBC9) and SUMO1, and modifies the substrate protein with SUMO1. We co-transformed the glutathione S-transferase (GST) tagged Src and pE1E2S1 in BL21, and antibiotic selection marker allows co-expression of pE1E2S1 and GST-Src of interest. As shown in Figure 1BL21. Bacteria lysates were used for GST pulldown, and immunoblotted with anti-SUMO1 and anti-Src antibodies (same membrane, stripped). (E) HEK293T cells were cotransfected with HA-c-Src, Flag-UBC9 and His-SUMO1 or SUMO2 or SUMO3. Cell lysates were treated with Ni2+-NTA resin, western blotting with anti-HA and anti-Src antibodies to confirm SUMO1 is the major SUMO modification. K318 is the Main SUMOylation Site of Src Next, we focus attention on the SUMOylation sites in Src protein. Based on the SUMOylation prediction software (

The photos of the colonies developed in soft agar were taken at day 21