Mouse islet isolation, cell lines, and tissue culture. All animals were used in accordance with The University of Chicago IACUC and ACUP protocol no. 71492. Islets were isolated from pancreata of 2- to 3-mo-old C57BL/6J wild-type mice (Jackson Laboratory, Bar Harbor, ME) using collagenase P (Roche Diagnostics, this Basel, Switzerland) digestion and a Ficoll gradient, as previously described (33). Islets were allowed to recover in RPMI 1640 medium supplemented with 10% FBS, 2 mM l-glutamine, 100 IU/ml penicillin, and 100 ��g/ml streptomycin and maintained in a humidified incubator at 37��C under an atmosphere of 95% air-5% CO2 and subsequently used for experiments. Similarly, islets from 10-wk-old Lepob/Lepob (ob/ob) males in the C57BL/6J background, and age-matched, sex-matched C57BL/6J islets were isolated and immediately analyzed by Western immunoblotting in three separate, independent experiments.
MIN6 cells were cultured in DMEM supplemented with 15% FBS, 10 IU/ml penicillin and 10 ��g/ml streptomycin and maintained in a humidified incubator at 37��C under an atmosphere of 95% air-5% CO2. All MIN6 cells were used between passages 20 and 35. Following transfection (24�C48 h), cells were either used for experiments or selected with an appropriate antibiotic. Zeocin and neomycin were purchased from Invitrogen and used at concentrations of 250 and 500 ��g/ml, respectively, in generation of stably transfected MIN6 cells. Stably transfected cell lines were of polyclonal origin. Quantitative real-time polymerase chain reaction.
Total RNA was extracted from MIN6 cells and freshly isolated C57BL/6J islets by use of the Tri Reagent method according to the manufacturer’s guidelines (Sigma-Aldrich). Total RNA (5 ��g) was reverse transcribed using SuperScript III (Invitrogen) and random hexamers per manufacturer’s protocol. Following cDNA synthesis, 50 ng of islet and MIN6 cell cDNA was analyzed for relative ERM message quantity using the FullVelocity SYBR Green QRT-PCR system (Stratagene) using primers designed against amplicons near the carboxyl terminus of the ERM cDNAs. Relative ERM expression in islets and MIN6 cells was assessed utilizing the 2?����CT method, as described (22), and normalized to ezrin (n = 3). SDS-PAGE and western immunoblotting. MIN6 cells and islets were maintained in KRBH with 2 mM glucose for 4 h in the dark, humidified, and at 37��C.
Subsequently, cells were stimulated with KRBH with high glucose (14 Anacetrapib mM islets and 20 mM MIN6 cells) for 10 min and immediately lysed in RIPA buffer containing 50 nM calyculin A (Sigma-Aldrich), 100 nM okadaic acid (Sigma-Aldrich), 1 mM AEBSF (Sigma-Aldrich), 1�� mammalian protease inhibitor cocktail (Sigma-Aldrich), and 1�� protein phosphatase inhibitor II cocktail (EMD Chemicals, Gibbstown, NJ) and quickly frozen in a dry ice-ethanol bath.