Ng regular medication. Height (metres) and weight (kilograms) were measured to determine the body mass index (BMI). The cohort was recruited to include four groups of individuals: seven of normal weight (BMI, 20?4 kg/m2), five overweight (BMI 25?9) and ten obese (BMI 30 kg/m2). The subjects were not habitually active. They were instructed to adhere to their usual diet and to refrain from any strenuous physical activity for 2 days before the study.Study protocolAll subjects had a screening visit to assess family history of diabetes and to confirm normal glucose tolerance with a standard (75 g of powdered glucose) 2-hour oral glucose tolerance test. Those individuals with a first-degree relative known to have diabetes or with diabetes according to the World Health Organisation diagnostic criteria (fasting glucose 7.0 mmol/l or 2 h glucose 11.1 mmol/l) were excluded from the study. Body composition (fat mass, fat free mass and bone mineral content) was determined by dual energy photon X-ray absorptiometry (DEXA; HOLOGIC Discovery W, Bedford, MA, version 12.1). Eligible subjects attended the Clinical Investigation Unit, Ninewells Hospital at 08:00 having not eaten for 12 hours, for a study protocol involving skeletal muscle biopsies and a hyperinsulinaemic, euglycaemic clamp [15]. A cannula was introduced into a forearm vein for infusion of insulin (Actrapid, NovoNordisk Copenhagen, Denmark) and 20 dextrose while on the contradetailed previously[11]. In brief, muscle biopsies were thawed in ice and homogenized (Dounce, 10?5 strokes) in 0.5 ml ice-cold lysis buffer (25 mM Tris-HCl (pH 7.4), 50 mM NaF, 100 mM NaCl, 1 mM sodium vanadate, 5 mM EGTA, 1 mM EDTA, 1 (v/v) Triton X-100, 10 mM sodium pyrophosphate, 0.27 M sucrose, Complete Protease inhibitor cocktail tablets (1 tablet/ 10 ml), and 0.1 (v/v) 2-mercaptoethanol). Protein lysates were obtained from the supernatant fraction after 10 min centrifugation at 13,000 r.p.m., and then pre-cleared for 1 h at 4uC with Protein G-Sepharose in PBS 50 (v/v). This process removed contaminating purchase 10236-47-2 antibodies present in the samples due to excess blood in the muscle samples. We found this step to be vital for consistent results with the subsequent immunoblot and immunoprecipitation steps. Western Blot Analysis. Protein extracts (30?0 mg protein) were separated on Novex SDS 4?2 polyacrylamide gels. Following transfer to nitrocellulose membranes, blots were first blocked with 5 (w/v) non-fat milk in TBST (Tris-buffered saline containing 0.1 (v/v) Tween 20) for 1 h, and incubated with primary antibodies at 4uC overnight prior to incubation for 1 h at room temperature with the secondary antibody and, finally development was carried out using an enhanced chemiluminescence (ECL) kit (Amersham Biosciences, Inc.). Bands obtained were quantified by densitometry using Scion Image software. Preand post-insulin protein lysates from each individual were run on gels as pairs, with 4 volunteers per gel, along with a buy AKT inhibitor 2 gel-to-gel normalisation control (volunteer 14, BMI of 24). Densitometry readings for every volunteer could then be normalised to volunteer 14 for comparison of either basal or post-insulin data. However this normalisation was not required when assessing response to insulin as this was done within a single immunoblot, after normalisation to the appropriate loading control (phosphorylation normalised to expression of same protein but on a separate gel using same sample run at same time rather than follo.Ng regular medication. Height (metres) and weight (kilograms) were measured to determine the body mass index (BMI). The cohort was recruited to include four groups of individuals: seven of normal weight (BMI, 20?4 kg/m2), five overweight (BMI 25?9) and ten obese (BMI 30 kg/m2). The subjects were not habitually active. They were instructed to adhere to their usual diet and to refrain from any strenuous physical activity for 2 days before the study.Study protocolAll subjects had a screening visit to assess family history of diabetes and to confirm normal glucose tolerance with a standard (75 g of powdered glucose) 2-hour oral glucose tolerance test. Those individuals with a first-degree relative known to have diabetes or with diabetes according to the World Health Organisation diagnostic criteria (fasting glucose 7.0 mmol/l or 2 h glucose 11.1 mmol/l) were excluded from the study. Body composition (fat mass, fat free mass and bone mineral content) was determined by dual energy photon X-ray absorptiometry (DEXA; HOLOGIC Discovery W, Bedford, MA, version 12.1). Eligible subjects attended the Clinical Investigation Unit, Ninewells Hospital at 08:00 having not eaten for 12 hours, for a study protocol involving skeletal muscle biopsies and a hyperinsulinaemic, euglycaemic clamp [15]. A cannula was introduced into a forearm vein for infusion of insulin (Actrapid, NovoNordisk Copenhagen, Denmark) and 20 dextrose while on the contradetailed previously[11]. In brief, muscle biopsies were thawed in ice and homogenized (Dounce, 10?5 strokes) in 0.5 ml ice-cold lysis buffer (25 mM Tris-HCl (pH 7.4), 50 mM NaF, 100 mM NaCl, 1 mM sodium vanadate, 5 mM EGTA, 1 mM EDTA, 1 (v/v) Triton X-100, 10 mM sodium pyrophosphate, 0.27 M sucrose, Complete Protease inhibitor cocktail tablets (1 tablet/ 10 ml), and 0.1 (v/v) 2-mercaptoethanol). Protein lysates were obtained from the supernatant fraction after 10 min centrifugation at 13,000 r.p.m., and then pre-cleared for 1 h at 4uC with Protein G-Sepharose in PBS 50 (v/v). This process removed contaminating antibodies present in the samples due to excess blood in the muscle samples. We found this step to be vital for consistent results with the subsequent immunoblot and immunoprecipitation steps. Western Blot Analysis. Protein extracts (30?0 mg protein) were separated on Novex SDS 4?2 polyacrylamide gels. Following transfer to nitrocellulose membranes, blots were first blocked with 5 (w/v) non-fat milk in TBST (Tris-buffered saline containing 0.1 (v/v) Tween 20) for 1 h, and incubated with primary antibodies at 4uC overnight prior to incubation for 1 h at room temperature with the secondary antibody and, finally development was carried out using an enhanced chemiluminescence (ECL) kit (Amersham Biosciences, Inc.). Bands obtained were quantified by densitometry using Scion Image software. Preand post-insulin protein lysates from each individual were run on gels as pairs, with 4 volunteers per gel, along with a gel-to-gel normalisation control (volunteer 14, BMI of 24). Densitometry readings for every volunteer could then be normalised to volunteer 14 for comparison of either basal or post-insulin data. However this normalisation was not required when assessing response to insulin as this was done within a single immunoblot, after normalisation to the appropriate loading control (phosphorylation normalised to expression of same protein but on a separate gel using same sample run at same time rather than follo.
