LY 249002, UO126, SP600125 and AG490 thirty minutes just before EGF treatment then harvested for Western blot on the instances indicated. Pancreatic islets had been isolated from 8 week outdated C57 Bl6 mice, as described previously, The islets were sepa rated by gradient centrifugation with Histopaque 1077 then handpicked under a dissection microscope and recovered in RPMI1640 media containing 10% FBS overnight. The next day, one hundred to 500 islets have been placed in 35 mm dishes containing RPMI1640 media with no FBS. After overnight starvation, islets had been trea ted for two h with EGF then harvested for RNA. These experiments had been approved through the Institu tional Animal Care Board at Rhode Island Hospital. Western blotting and immunoprecipitation Entire cell lysates have been collected in RIPA buffer for Western blot analyses.
50 ug of protein per lane was separated on a 12 15% SDS Page. Gels were trans ferred to nitrocellulose membranes then blotted by using a rabbit anti survivin antibody in 5% non extra fat dry milk, fol lowed by anti rabbit antibody, Nuclear extracts from INS 1 cells were pre pared according on the system of Schreiber et al, Total protein concentration was measured utilizing selleck chemicals BCA protein assay kit towards a bovine serum albumin regular curve. Cells were collected, washed with PBS twice and pelleted by centrifugation. The cell pellet was resuspended in 50 ul cold buffer A vortexed for 10 sec, then shaken on a rocker vig orously for ten 15 min. The lysate was centrifuged at 13000 RPM for 10 15 min. The supernatant containing cytoplasm was transferred to a fresh tube.
The nuclear pellet was resuspended in 50 ul ice cold buffer C and the tube vigorously rocked at 4 C for 15 min on a shaking platform. The nuclear extract was selleck GSK2118436 centrifuged for ten min at 13000 RPM at four C as well as supernatant transferred to a brand new tube. Nuclear or cytoplasmic fractions have been resolved by SDS Web page. PVDF membranes have been probed with mouse anti survi vin, rabbit anti HDAC1 or mouse anti Actin, For the ubiquitin experiments, INS 1 cells have been pre handled with MG132 for three h just before becoming trea ted with EGF for one h. Lysates have been precleared with 15 ul of Protein A G PLUS Agarose for 1 h just before overnight incubation with one ug of either regular rabbit IgG or rabbit poly clonal Survivin key antibody. 30 ul of Protein A G Plus Agarose beads had been then additional to form immunocomplexes and samples were shaken for two h.
Samples were then spun at 3000 RPM for 5 min. Supernatant was discarded and Protein A G Plus Agarose beads were washed four instances and after that resuspended and boiled in the SDS loading buf fer. All previous procedures are performed at 4 C unless of course otherwise noted. Immunocomplexes were resolved by SDS Web page. PVDF membranes had been probed with mouse anti survivin or rabbit anti ubiquitin, Cyclohexamide remedy INS one cells were starved overnight then handled with or without having EGF to get a total of 4 hours.