phenylmethanesulfonyl fluoride

Conditions: temp:0.00; pressure:0.00; stir:

Conditions: temp:0.00; pressure:0.00; stir:

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: For each assay, an aliquot of receptor (1:1000-1:3000 dilution) was incubated in TEGM (10 mM Tris, pH 7.2, 1 mM EDTA, 10% glycerol, 7 μl/100 ml β-mercaptoethanol, 10 mM Na molybdate, 1 mM dithiothreitol, 5 μg/ml aprotinin, 2 μg/ml leupeptin, 2 μg/ml benzamide and 0.5 mM PMSF) containing 5-10% COS-1 cell cytoplasmic lysate and 10 nM labeled thiazolidinedione ([3H2 ]AD-5075, 21 Ci/mmole), ± test compound compound, [3H2 ]Example 11, 17 Ci/mmole), ± test compound, respectively. Assays were incubated for ~16 h at 4 ° C. in a final volume of 300 μl. Unbound ligand was removed by addition of 200 μl dextran/gelatin-coated charcoal, on ice, for ~10 minutes. After centrifugation at 3000 rpm for 10 min at 4° C., 200 μl of the supernatant fraction was counted in a liquid scintillation counter. In this assay the KD for AD-5075 and Example 11 is 1 nM, respectively. containing 5-10% COS-1 cell cytoplasmic lysate and 10 nM Assays were incubated for ~16 h at 4 ° C. Unbound ligand was removed by addition of 200 μl dextran/gelatin-coated charcoal, on ice, for ~10 minutes

Conditions: temp:0.00; pressure:0.00; stir:

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: After induction, the cultures were harvested by centrifugation at 5,000 rpm for ten minutes at 4° C. The cells were then resuspended in 1/100 volume of buffer A (100 mM potassium phosphate, pH 7.4, 0.5% sodium cholate, 20% glycerol, 0.1 mM EDTA, 0.1 mM DTT and 0.5 mM PMSF). The cells were then lysed in buffer A with 200 μg/ml lysozyme. The supernatant was collected after spinning down the total lysate at 100,000×g for one (1) hour at 4° C. The pellet was resuspended thoroughly in the same buffer and centrifuged again. Both supernatants were combined and stored on ice overnight after the addition of 100 units of DNaseI. The clear lysate was then applied to an Octylamino Sepharose 4B column (2.6×15 cm). This column was washed and eluted with the same buffer. The eluted fractions were dialyzed against buffer B (10 mM potassium phosphate, pH 7.4, 0.2% sodium cholate, 0.2% Emulgen 911, 0.1 mM EDTA, 0.05 mM DTT, 0.5 mM phenyl methyl sufonyl fluoride (PMSF)), applied to a hydroxyapatite column (2.4×7 cm) and equilibrated with the same buffer. This column was then washed with 200 ml of 10 mM potassium phosphate buffer, then with 150 ml of 50 mM potassium phosphate buffer and then eluted with 300 ml of 100 mM potassium phosphate buffer. These three buffers also contained 20% glycerol, 0.3% sodium cholate, 0.05 mM EDTA, 0.1 mM DTT and 0.5 mM PMSF. The purified sample was brought to 0.2% Emulgen 911 and then dialyzed against 10 mM potassium phosphate buffer, pH 7.4, 20% glycerol, 0.1 mM EDTA, 0.1 mM DTT, 0.5 mM PMSF and 0.2% Emulgen 911 (buffer C). The sample was then applied to a second hydroxylapatite column (0.5×3.0 cm) equilibrated with buffer C and eluted with buffer C but containing 360 mM potassium phosphate. At this stage, the purity of the human cholesterol 7α-hydroxylase was confirmed by SDS-polyacrylamide gel electrophoresis. After induction, the cultures were harvested by centrifugation at 5,000 rpm for ten minutes at 4° C The supernatant was collected after spinning down the total lysate at 100,000×g for one (1) hour at 4° C stored on ice overnight after the addition of 100 units of DNaseI This column was washed eluted with the same buffer The eluted fractions were dialyzed against buffer B (10 mM potassium phosphate, pH 7.4, 0.2% sodium cholate, 0.2% Emulgen 911, 0.1 mM EDTA, 0.05 mM DTT, 0.5 mM phenyl methyl sufonyl fluoride (PMSF)) This column was then washed with 200 ml of 10 mM potassium phosphate buffer with 150 ml of 50 mM potassium phosphate buffer and then eluted with 300 ml of 100 mM potassium phosphate buffer dialyzed against 10 mM potassium phosphate buffer, pH 7.4, 20% glycerol, 0.1 mM EDTA, 0.1 mM DTT, 0.5 mM PMSF and 0.2% Emulgen 911 (buffer C) eluted with buffer C containing 360 mM potassium phosphate

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: After induction, the cultures were harvested by centrifugation at 5,000 rpm for ten minutes at 4° C. The cells were then resuspended in 1/100 volume of buffer A (100 mM potassium phosphate, pH 7.4, 0.5% sodium cholate, 20% glycerol, 0.1 mM EDTA, 0.1 mM DTT and 0.5 mM PMSF). The cells were then lysed in buffer A with 200 μg/ml lysozyme. The supernatant was collected after spinning down the total lysate at 100,000×g for one (1) hour at 4° C. The pellet was resuspended thoroughly in the same buffer and centrifuged again. Both supernatants were combined and stored on ice overnight after the addition of 100 units of DNaseI. The clear lysate was then applied to an Octylamino Sepharose 4B column (2.6×15 cm). This column was washed and eluted with the same buffer. The eluted fractions were dialyzed against buffer B (10 mM potassium phosphate, pH 7.4, 0.2% sodium cholate, 0.2% Emulgen 911, 0.1 mM EDTA, 0.05 mM DTT, 0.5 mM phenyl methyl sufonyl fluoride (PMSF)), applied to a hydroxyapatite column (2.4×7 cm) and equilibrated with the same buffer. This column was then washed with 200 ml of 10 mM potassium phosphate buffer, then with 150 ml of 50 mM potassium phosphate buffer and then eluted with 300 ml of 100 mM potassium phosphate buffer. These three buffers also contained 20% glycerol, 0.3% sodium cholate, 0.05 mM EDTA, 0.1 mM DTT and 0.5 mM PMSF. The purified sample was brought to 0.2% Emulgen 911 and then dialyzed against 10 mM potassium phosphate buffer, pH 7.4, 20% glycerol, 0.1 mM EDTA, 0.1 mM DTT, 0.5 mM PMSF and 0.2% Emulgen 911 (buffer C). The sample was then applied to a second hydroxylapatite column (0.5×3.0 cm) equilibrated with buffer C and eluted with buffer C but containing 360 mM potassium phosphate. At this stage, the purity of the human cholesterol 7α-hydroxylase was confirmed by SDS-polyacrylamide gel electrophoresis. After induction, the cultures were harvested by centrifugation at 5,000 rpm for ten minutes at 4° C The supernatant was collected after spinning down the total lysate at 100,000×g for one (1) hour at 4° C stored on ice overnight after the addition of 100 units of DNaseI This column was washed eluted with the same buffer The eluted fractions were dialyzed against buffer B (10 mM potassium phosphate, pH 7.4, 0.2% sodium cholate, 0.2% Emulgen 911, 0.1 mM EDTA, 0.05 mM DTT, 0.5 mM phenyl methyl sufonyl fluoride (PMSF)) This column was then washed with 200 ml of 10 mM potassium phosphate buffer with 150 ml of 50 mM potassium phosphate buffer and then eluted with 300 ml of 100 mM potassium phosphate buffer dialyzed against 10 mM potassium phosphate buffer, pH 7.4, 20% glycerol, 0.1 mM EDTA, 0.1 mM DTT, 0.5 mM PMSF and 0.2% Emulgen 911 (buffer C) eluted with buffer C containing 360 mM potassium phosphate

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Sf21 cells were infected with a baculovirus expression vector for BTK or SYK, as previously described (refs 18 and 19). Cells were harvested, lysed (10 mM Tris pH 7.6, 100 mM NaCl, 1% Nonidet P-40, 10% glycerol, 50 mM NaF, 100 mM Na3VO4, 50 μg/ml phenylmethylsulfonyl fluoride, 10 μg/ml aprotonin, 10 μg/ml leupeptin), and the kinase proteins were purified to homogeneity, as reported (refs 18 and 19). the kinase proteins were purified to homogeneity

Conditions: temp:96.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: RINm5F cells following different treatments were lysed in a lysis buffer (pH 7.5) consisting of 50 mM HEPES, 150 mM NaCl, 1 EGTA, 1 mM EDTA, 10% glycerol, 1% triton X-100, 1 mM PMSF and a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The lysate was centrifuged at 800×g for 10 min at 4° C. to remove cell debris and nuclei. The protein concentration of the resulting samples was determined with Bio-Rad protein assay reagent (Bio-Rad, Hercules, Calif.). The samples were then denatured by heating at 96° C. for 3 min in SDS sample buffer and underwent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis. Briefly, 50, 90 or 180 μg proteins were separated in discontinuous gels consisting of a 4% acrylamide stacking gel (pH 6.8) and an 8% acrylamide separating gel (pH 8.8). The separated proteins were then electroblotted to hydrophobic polyvinylidene difluoride membrane (Hybond-P; GE Healthcare, Uppsala, Sweden). The blots were blocked by incubation for 1 h with 5% non-fat milk powder in a washing buffer, containing 50 mM tris(hydroxymethyl)aminomethane, 150 mM NaCl and 0.05% Tween 20 (pH 7.5). They were then incubated overnight at 4° C. with affinity-purified rabbit polyclonal antibodies to β1 integrin (1:500; Millipore, Billerica, Mass.), CaV1.2 (1:200) and CaV1.3 (1:200), respectively, and for 1 h at room temperature with mouse monoclonal antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:4000; Applied Biosystems/Ambion, Austin, Tex.), respectively. After rinsing with the washing buffer, the blots were incubated with the secondary antibodies (either horseradish peroxidase-conjugated goat anti-rabbit IgG or horseradish peroxidase-conjugated goat anti-mouse IgG; 1:50,000; Bio-Rad, Hercules, Calif.) at room temperature for 45 min. The immunoreactive bands were visualized with the ECL plus Western blotting detection system (GE Healthcare, Uppsala, Sweden). to remove cell debris Briefly, 50, 90 or 180 μg proteins were separated in discontinuous gels The blots were blocked by incubation for 1 h with 5% non-fat milk powder in a washing buffer, containing 50 mM tris(hydroxymethyl)aminomethane, 150 mM NaCl and 0.05% Tween 20 (pH 7.5) They were then incubated overnight at 4° C. with affinity-purified rabbit polyclonal antibodies to β1 integrin (1:500; Millipore, Billerica, Mass.), CaV1.2 (1:200) and CaV1.3 (1:200) respectively, and for 1 h at room temperature with mouse monoclonal antibody to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:4000; Applied Biosystems/Ambion, Austin, Tex.) After rinsing with the washing buffer, the blots were incubated with the secondary antibodies (either horseradish peroxidase-conjugated goat anti-rabbit IgG or horseradish peroxidase-conjugated goat anti-mouse IgG; 1:50,000; Bio-Rad, Hercules, Calif.) at room temperature for 45 min

Conditions: temp:-80.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Fresh intact proximal jejunum was removed, washed with saline and placed on ice while approximately 4 g of mucosa were removed and transferred to cold 2 mM Tris-HCl buffer (pH 7.1) containing 50 mM mannitol and protease inhibitors (5 μM aprotinin, leupeptin, and pepstatin). The mucosa was then homogenized and PEG 4000 was added to a final concentration of 10% and stirred on ice for 15 minutes. The homogenate was then centrifuged for 15 minutes at 7,500×g and the resulting supernatant fraction centrifuged at 27,000×g for 60 minutes at 4° C. The pellet was washed in suspension buffer (10 mM Tris-HCl, pH 7.1, containing 300 mM mannitol and protease inhibitors 5 μM aprotinin, leupeptin, and pepstatin) and collected again by centrifugation for 5 minutes, 27,000×g at 4° C. The crude brush border membrane (BBM) pellet was suspended in 1 mL of suspension buffer. For preparation of total membranes, frozen jejunum sections (1 g) were and homogenized on ice in 700 μL Buffer A (50 mM Tris-HCl pH 7.5, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 10% glycerol) containing 1% Triton X-100 and 5 μM aprotinin, leupeptin, and pepstatin. The homogenates were centrifuged at 6,000×g for 20 minutes at 4° C. to remove insoluble material. The protein concentrations of the total and BBM preparations were determined using BCA reagents (Pierce, Rockford, Ill., USA). Final total and brush border membrane preparations were frozen at −80° C. until assayed. The purity of the brush border membrane preparations as measured by alkaline phosphatase were not affected by treatment (data not shown). Fresh intact proximal jejunum was removed washed with saline were removed containing 50 mM mannitol and protease inhibitors (5 μM aprotinin, leupeptin, and pepstatin) PEG 4000 was added to a final concentration of 10% stirred on ice for 15 minutes g for 60 minutes at 4° C The pellet was washed in suspension buffer (10 mM Tris-HCl, pH 7.1 containing 300 mM mannitol and protease inhibitors 5 μM aprotinin leupeptin, and pepstatin) and collected again by centrifugation for 5 minutes g at 4° C For preparation of total membranes frozen jejunum sections (1 g) g for 20 minutes at 4° C. to remove insoluble material

Conditions: temp:-80.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Fresh intact proximal jejunum was removed, washed with saline and placed on ice while approximately 4 g of mucosa were removed and transferred to cold 2 mM Tris-HCl buffer (pH 7.1) containing 50 mM mannitol and protease inhibitors (5 μM aprotinin, leupeptin, and pepstatin). The mucosa was then homogenized and PEG 4000 was added to a final concentration of 10% and stirred on ice for 15 minutes. The homogenate was then centrifuged for 15 minutes at 7,500×g and the resulting supernatant fraction centrifuged at 27,000×g for 60 minutes at 4° C. The pellet was washed in suspension buffer (10 mM Tris-HCl, pH 7.1, containing 300 mM mannitol and protease inhibitors 5 μM aprotinin, leupeptin, and pepstatin) and collected again by centrifugation for 5 minutes, 27,000×g at 4° C. The crude brush border membrane (BBM) pellet was suspended in 1 mL of suspension buffer. For preparation of total membranes, frozen jejunum sections (1 g) were and homogenized on ice in 700 μL Buffer A (50 mM Tris-HCl pH 7.5, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 10% glycerol) containing 1% Triton X-100 and 5 μM aprotinin, leupeptin, and pepstatin. The homogenates were centrifuged at 6,000×g for 20 minutes at 4° C. to remove insoluble material. The protein concentrations of the total and BBM preparations were determined using BCA reagents (Pierce, Rockford, Ill., USA). Final total and brush border membrane preparations were frozen at −80° C. until assayed. The purity of the brush border membrane preparations as measured by alkaline phosphatase were not affected by treatment (data not shown). Fresh intact proximal jejunum was removed washed with saline were removed containing 50 mM mannitol and protease inhibitors (5 μM aprotinin, leupeptin, and pepstatin) PEG 4000 was added to a final concentration of 10% stirred on ice for 15 minutes g for 60 minutes at 4° C The pellet was washed in suspension buffer (10 mM Tris-HCl, pH 7.1 containing 300 mM mannitol and protease inhibitors 5 μM aprotinin leupeptin, and pepstatin) and collected again by centrifugation for 5 minutes g at 4° C For preparation of total membranes frozen jejunum sections (1 g) g for 20 minutes at 4° C. to remove insoluble material

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Culture supernatants were sampled at approximately 50 and 100 hours, treated with 2 mM PMSF final concentration, and frozen. To estimate NOVAMYL™ expression, supernatants were mixed with an equal volume of 2X Laemmli loading buffer, immediately boiled, and loaded on 14% or 8-16% acrylamide TRIS-glycine gels purchased from a commercial source (NOVEX, San Diego, Calif.). Known amounts of a NOVAMYL™ standard were also loaded on the same gel to estimate the amount of NOVAMYL™ produced. In some cases, NOVAMYL™ titer was determined using maltotriose as substrate. Specifically, a sample of the enzyme is incubated with maltotriose at pH 5.0 and 37° C. for 30 minutes. The reaction is then stopped by adjusting the pH to approximately 11. The amount of glucose produced from the breakdown of maltotriose to glucose and maltose is then measured with glucose dehydrogenase and NADH at 340 nm under standard conditions. Known amounts of a NOVAMYL™ standard (Novo Nordisk A/S, Bagsvaerd, Denmark) are also run. Culture supernatants were sampled at approximately 50 and 100 hours concentration frozen were mixed with an equal volume of 2X Laemmli loading buffer produced

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Culture supernatants were sampled at approximately 50 and 100 hours, treated with 2 mM PMSF final concentration, and frozen. To estimate NOVAMYL™ expression, supernatants were mixed with an equal volume of 2X Laemmli loading buffer, immediately boiled, and loaded on 14% or 8-16% acrylamide TRIS-glycine gels purchased from a commercial source (NOVEX, San Diego, Calif.). Known amounts of a NOVAMYL™ standard were also loaded on the same gel to estimate the amount of NOVAMYL™ produced. In some cases, NOVAMYL™ titer was determined using maltotriose as substrate. Specifically, a sample of the enzyme is incubated with maltotriose at pH 5.0 and 37° C. for 30 minutes. The reaction is then stopped by adjusting the pH to approximately 11. The amount of glucose produced from the breakdown of maltotriose to glucose and maltose is then measured with glucose dehydrogenase and NADH at 340 nm under standard conditions. Known amounts of a NOVAMYL™ standard (Novo Nordisk A/S, Bagsvaerd, Denmark) are also run. Culture supernatants were sampled at approximately 50 and 100 hours concentration frozen were mixed with an equal volume of 2X Laemmli loading buffer produced