ANÁLISIS DEL CONCEPTO DE CONTRIBUYENTE NO

(a) In vitro kinase assays P K B, PllO, A-Kinase

W hen assaying im m unoprecipitates they were w ashed twice in lysis buffer and twice in assay buffer. Protein kinase assays w ere generally carried out in a reaction volum e of 50pl. To m easure pllO protein kinase activity the buffer used contained; 50mm HEPES, pH7.4, 150mM NaCl, lOmM M nClz. For all other kinases activity was m easured in lOOmM Tris-HCl, pH7.5 and 20mM MgClj. Reactions w ere begun by the addition of lOOpM ATP (0.5uCi of [/^P]ATP) follow ed by 20 m inutes incubation at room tem perature and stopping by the addition of lOul of WLD. Samples were ru n on SDS-polyacrylamide gels, the gels were fixed and dried (see2.2.3.5), and the products of the kinase reactions were visualised by autoradiography.

(b) Phosphocellulose cation-exchange paper assay

Phosphocellulose p ap er w as used for m easuring the p h o sp h o ry la tio n of synthetic peptides by protein kinases. P81 cation exchange paper w as used so that the basic residues of peptides can bind at low pH while free [y-^^P] ATP does not, and can be w ashed away.

To m easure p i 10 protein kinase activity the buffer u sed contained; 50mm HEPES, pH7.4, 150mM NaCl, lOmM MnClz . For all other kinases activity w as m easured in lOOmM Tris-HCl, pH7.5 and 20mM MgClz. The peptides (0.5mM) w ere mixed w ith assay buffer and enzyme in a total volum e of 45pl before the addition of 5|il lOOjiM ATP (0.5|xCi of [y^^P]ATP) to each tube w ith 20 second intervals. Samples were vortexed and placed in a w ater bath set at 30°C. After 20 m inutes a 30jil aliquot was removed from each tube, at 20 second intervals, and spotted on to a 2x2 cm P81 paper square and dro p p ed into a w ire cage in a 500ml beaker, containing approxim ately 200ml 75mM orthophosphoric acid. W hen all reactions were finished the first w ash was p o ured into a radioactive w a ste co n ta in e r an d 3 m ore w ash es w ere c arried o u t w ith 75mM orthophosphoric acid (500ml per w ash for 5minutes). Papers were w ashed once w ith ethanol before being dryed. Papers were then counted in a scintillation counter in 10ml organic scintillant.

2.2A.2 PI3-kinase assay

(a) A ssa y procedure

Cells were stim ulated, lysed and im m unoprecipitated, as described previously, u sin g an tiphosphotyrosine antibody (1:100). The assay w as p erfo rm ed as

described previously (Nave et al., 1996). The beads were w ashed twice in lysis buffer an d twice in 2X kinase assay buffer (100mm HEPES, p H 7.4, 200mM NaCl, 2mM DTT) excess buffer w as rem oved using a ham ilton syringe and resuspended in 25pl 2X assay buffer and lOul PI (Im g /m l) and incubated on ice for 10 m inutes. 15pl A T P/M g mix (lOOpM ATP, IpC i [yP]ATP, 5mM MgClz ) w as then ad d ed and incubated for 20 minutes at room tem perature. The reaction w as te rm in a te d by the a d d itio n of lOOpl of 0.1 M H C l an d 200|xl of c h lo ro fo rm /m e th a n o l (1:1). The m ixture w as vortexed and centrifuged at 10,000rpm for 2 m inutes at room tem perature. The upper phase w as discarded and the low er organic phase w ashed w ith 80pl m ethanol, IM HCl (1:1). After cen trifu g atio n the u p p e r phase w as again discarded an d the low er phase evaporated to dryness.

(b) A n a lytica l methods

D ried phospholipids w ere resuspended in 30|il chlorofom /m ethanol (4:1) and sp o tted onto a Silica Gel-60 coated alum inium backed 2 0x2 0cm TLC plates (Merck, 5554). The TLC plates had been pretreated by dipping into a solution of 1% oxalic acid, Im M EDTA in w ater/ m ethanol (6:4) and baking at 150°C for 2 h ours. TLC plates w ere developed in c h lo ro fo rm /m e th a n o l/4M am m onia (9:7:4).

2.2A.3 HSL / neutral cholesterol ester a ctivity assay

(a) Cell preparation. To m easure the neutral cholesterol esterase activity in m acrophage lysates the cell m onolayers w ere w ashed w ith PBS before being scraped into 200ul of assay buffer (5mM imidazole pH 7.0, 30% glycerol, 50mM

NaCl, 20mM EDTA, 5mM sodium pyrophosphate, O.lmM benzam idine, Im M DTT, 5 p g /m l leupeptin, Ip g /m l pepstatin, 200KIU aprotinin). Cells were lysed w ith 4 rounds of freeze-thawing followed by centrifugation at 14000rpm at 4°C. 25|il aliquots of the cell lysate (approximately 0.15 mg protein) w ere assayed. To directly m easure HSL activity the pro tein w as first isolated from cells by im m u n o p ré c ip ita tio n w ith an ti-H S L a n tib o d y (see 2.2.3.2). The im m unoprecipitate w as w ashed 2 times in lysis buffer follow ed by 2 w ashes w ith assay buffer. All buffer was rem oved w ith a H am ilton syringe before the im m unprecipitate was resuspended in 25|il of assay buffer.

{h)Substrate preparation and assay procedure. The assay w as perform ed as previously described (Khoo et al., 1976). Briefly, the substrate (3.5ml volum e, e n o u g h for 19 assays) is p re p a re d b y d ry in g d o w n a m ix tu re of Cholestero^^'^Cjoleate (2.5|xCi, 30pg) and cholesterol oleate (400|ig) u n d e r nitrogen. The lipid w as then resuspended in lOOul ethanol and added to; lOOmg BSA (sigma A60093), 1ml 0.2M sodium phosphate buffer, pH 7 an d 2.4 ml distilled w ater in a flask which was being sw irled swiftly in constant m otion at room tem perature. 175pl substrate was added to 25|il sam ple and incubated for 50 m inutes at 37°C. To stop the reaction 0.5ml of stopping buffer w as added (O.IM potassium tetraborate,0.1M potassium carbonate, p H 10.5) follow ed by 1.6 m l extraction buffer (m ethanol (1.41)/ chloroform (1.25)/ h e p tan e(l)) containing 0.3% (w /v) of unlabeled oleic acid as carrier. The m ixture w as vortexed vigorously and then centrifuged at 3000rpm for 10 m inutes at 10°C. 0.5ml of the u p p er layer containing [^^C]oleic acid w as transferred to vials containing 5 ml of scintillation fluid. 1 u n it of enzym e activity catalyses the

release of IpM ole oleate per minute, (see figures 2.1, 2.2, 2.3. for characterisation of assay)

2.2A.4 Cholesterol cell loading assay

(a) Lipoprotein preparation:

Lipoproteins were isolated by density gradient ultracentrifugation in a vertical rotor, as previously described (Chung 1980, C hung 1986). LDL (1ml in the range of 8-16 m g /m l) was acetylated by the addition of 1ml saturated sodium acetate solution followed by the addition of acetic anhydride (Basu, 1976) (at a volum e in ul 1.5 times the num ber of m g LDL protein). Acetic anhydride w as ad d ed in 2-4 ul aliquots over an hour, then incubated for a further 30 m inutes at 4°C. A cétylation of lipoprotein w as confirm ed by loading sam ples on 0.85-1% agarose gel at p H8.6 (acetylated LDL ru n s faster th an u n m o d ified LDL). A cetylated LDL was then de-salted using PDIO colum ns (Pharmacia, 17-0851- 01) and concentrated (vivaspins, vivascience VS 1521) before filter sterilising.

(h) Freezing o f lipoproteins

C ryopreservation of LDL in 10% sucrose (w /v ) has previously been show n to prevent structural and functional changes. Sucrose was added by direct addition of a stock solution (50% sucrose, 150mm NaCl, 0.24mM EDTA, pH7.4). Prior to all experim ental procedures, sucrose w as rem opved by dialysis at 4°C against saline (150mMNaCl, 0.24mM EDTA, pH7.4). Dialysis buffer w as changed 3 times over a 24 hour period.

(c) Preparation ofP^C] oleate label

12g of BSA (fatty acid free) w as added to 35 ml of solution 1. (150mm NaCl, 50mM Tris-HCl pH7.4) in 2g aliquots over a 5 hour period, stirring continuously at room tem perature. The pH was adjusted to 7.4 and the volum e m ade up to 50ml w ith solution 1, this is solution 2 (24% BSA solution). To make solution 3 (sodium oleate complexed w ith 12% BSA), 90mg of oleic acid w as dissolved in 2 ml ethanol and lOOjil 5M NaOH. The lipid was then dried u n d er nitrogen and then 10 ml of solution 1 w as added. This was heated to 60°C for 3-5 m inutes before addition of 12.5ml ice-cold solution 2 and stirring for 10 m inutes at room tem perature. The final volum e was adjusted to 25ml w ith solution 1. Finally [^^C]oleate-0.12% BSA w as prepared by drying 250|iCi [^^Cjoleic acid (specific activity 50-60mCi/mMol) u nder nitrogen and resuspending in 4.35 m l solution 3. 0.8ml solution 2 w as ad d ed followed by 0.8ml solu tio n l. and w as stirred gently for 4-6 hours at room temperature. The solution was stored at -20oC.

(d) Cell Incubations:

Cell incubations and analysis was carried out as described previously ( Graham et al., 1996). Cells were plated into 6-well (35mm) plates. Prior to loading cells were incubated overnight in RPMI 1640 m edium containing 2 m g /m l BSA and no serum . Cells w ere w ashed twice in PBS before the a d d itio n of RPMI containing the relevant am ount of glucose, insulin/leptin, lOOpg/ml acetylated LDL and 2pC i/m l [^^Cjoleate. Cells were then incubated for 24 hours.

(e) A nalytical methods:

Cells w ere w ashed twice in PBS and h arv ested into 0.5ml PBS. 0.5ml of chloroform /m ethanol (2:1) was added followed by vortexing for 10 seconds before centrifugation at 1800g for 10 m inutes at 10°C. The lower organic phase w as tran sferred to a glass vial an d the u p p e r phase w as w ash ed w ith chloroform /m ethanol as before. Again the low er organic phase was transferred to the glass vial. Chloroform was rem oved by drying u n d er nitrogen. Lipids were resuspended in isopropanol and separated by thin layer chrom atography on silica gel plates developed in hexane/ethyl ether/acetic acid (80:20:1 v /v /v ) . Radioactive bands w ere detected using a Fuji FLA2000 phosphoim ager and were quantitated.