Page 67 - 76_03

VOL. 76 (3), 357-377, 2010 TSC1-TSC2 COMPLEX ON THE CROSSROAD OF PANCREATIC...

2.2. Origin of pancreatic ß cell lines and cell culture

IR +/+ and IR -/- pancreatic ß cells from IR loxP mice were gener-
ated as described before (10). From IR -/- cells, expression of a single
IR isoform was reconstituted by retroviral infection, and the differen-
tial mRNA splicing was assessed by RT-PCR as previously described
(27). TSC2 -/- and TSC2 +/+ MEFs were provided by D. J. Kwiatkowsky
(Dana-Farber Cancer Institute, Boston, MA).

2.3. Cell signalling, Western-blotting and immunofluorescence
assays

For cell signaling experiments, cells were serum and glucose
starved for 3 h in DMEM containing 0.2 mM glucose and 0.5% bovine
serum albumin (BSA), and subsequently stimulated with insulin 10
nM or glucose 5 mM for 5 or 15 min respectively. Inhibitors were
added 30 min prior to stimulus. Cells were washed and lysed for pro-
tein extraction, protein concentration was determined and samples
were subjected to SDS-PAGE for Western-blot according to standard
procedures (10). Immunofluorescence, cell cycle and violet crystal as-
says assays were carried as decribed previously (10).

2.4. siRNA trasnfection

siRNA against the expression of mouse TSC2 was designed and
chemically synthesized by Bionova Científica SL. For siRNA transfec-
tion, cells were electroporated using Nucleofector II and Cell Line Kit
T (Amaxa) following the instructions from the manufacturer. A pool
of three different siRNA (2 µg) was added to the transfection reagent
per 2x106 cells. After transfection, cells were seeded and protein ex-
pression was checked after 24-48 h

2.5. ?-Phosphatase treatment

100 µg of total lysates were mixed in ?-Phosphatase Buffer (50 mM
Tris-HCl pH7.5, 0.1 mM Na2EDTA, 5 mM dithiothreitol, 0.01% BRIJ

361