RESULTADOS RESPECTO AL MINISTERIO DE EDUCACIÓN

0.25 g of protein A sepharose (CL4B - Sigma) were weighed out and washed with 3 changes of PBS to make 1 ml of swollen beads (1 ml of beads has a binding capacity of 5 mg human IgG). 1 ml of Hoxie 21 ascites (26.29 mg/ml) was adjusted to pH 9.00 and 3 M NaCl, and incubated with the beads for 2 h at room tem perature with gentle mixing. The m ixture was centrifuged at 1200 g for 5 m in , the su p e rn a ta n t re m o v e d , and its p ro tein c o n c e n tr a tio n determined at 280 nm in a spectrophotometer (LKB Ultrospec HE). The beads were washed twice with 10 volumes of 3 M NaCl/0.05 M N a2B4 0 7. I O H2O, and resuspended in 10 ml 3 M N aC l/0.2 M N a2B4 0? .1 0H2 0 containing 20 mM dim eth y lp im elim id ate (Pierce and W arrin e r) fo r 30 m in at ro o m te m p e ra tu re w ith g en tle m ixing. The m ixture was centrifuged at 1200 g for 5 m in, the supernatant removed and the beads were washed once with 0.2 M e th a n o la m in e (p H 8.00). I n c u b a te d the b e ad s in 0 .2 M ethanolam ine (pH 8.00) for 2 h at room tem perature with gentle mixing. The beads were washed twice with PBS and stored in PB S/0.02% NaNg at 4°C. These anti-CD4 protein A sepharose beads were tested for quantitative precipitation o f CD4. One 100 mm plate of HeLa-CD4 cells was washed with PBS and scraped into 10

ml PBS at 4°C. The cell suspension was centrifuged at 300 g for 5 min at 4°C and the cell pellet was resuspended in 200 p i Tris lysis buffer pH 8.00, containing 3% NP40, 150 mM NaCl, 2 mM EDTA and p ro tea se inhibitors (1 mM P M SF and 10 p g / m l each of chym ostatin, leupeptin, antipain and pepstatin) for 15 min on ice. T he detergent-insoluble m aterial was rem oved by centrifuging at full speed (13 000 rpm) in a m icrofuge (Heraeus) for 20 min at 4°C. The supernatant was recovered, and 15 pi, 20 pi, and 25 p i of p rew ash ed anti-CD4 beads were added to 50 p i aliquots of the HeLa-CD4 lysate for 1 h at 4°C with gentle mixing. The beads were washed 3 times with TBS/0.5% TXlOO and once with TBS to red u ced the d etergent co n ten t o f the sam ples. Each p ellet of beads was resuspended into 50 p i non-reducing 1 x sample buffer. Added 5 p i of non-reducing, 5 x sample buffer to 20 p i aliquots of the supernatants after im m u n o p récip itatio n , and 2 0 p i of lysate before precipitation. All samples were and heated to 9 5 °C for 5 min, then loaded onto 1 0% acrylamide gels which were run at 2 0 m A for approxim ately 1 h. T he proteins were transferred to n itr o c e llu lo s e fo r 30 min at 1 A and im m u n o b lo tte d (see immunoblotting below) for CD4.

2.5 D e te c tio n O f C ell S u r fa c e C D 4 A ft e r P M A T rea tm e n t, Cells in 16 mm diameter tissue culture wells were washed twice w ith b in d in g m ed iu m (BM: R P M I 1640 lack in g b ica rb o n ate, supplemented with 0.2% BSA, and 10 mM HEPES, pH 7.40) at 37°C, b efo re incu b atio n in BM in the presen ce of 100 ng/m l PM A (Sigma) for times up to 6 h. Cells were cooled quickly by washing twice with BM at 4°C and incubated with gentle shaking in BM containing 0.3 nM ^:^^I-Q4120 for 2 h at 4°C . Unbound antibody was removed by washing 3 times with BM and 2 washes with PBS at 4°C. The cells were harvested in 400 p i 0.2 M NaOH and the wells rinsed with 400 p i H2O w hich was then pooled with the respective cell lysate. The lysates were counted in the gamma c o u n te r and the level of an tibody bound for each time p o in t determ ined as a proportion of the original am ount of antibody bound at the cell surface before the addition of PMA.

2.6 Detection O f Cell Surface CD4 A fte r PMA Treatment In Hyp ertonic Medium,

HeLa-CD4 cells grown for 3 days in 16 m m diameter wells were preincubated for 5 min at 4°C in in the presence or absence o f h y p e rto n ic m e d iu m (0.45 M su c ro se in R P M I 1640 lac k in g bicarbonate, supplemented with 0.2% BSA, 20 m M MBS, and 20 m M su ccin ic acid, pH 5.70), and w arm ed in fresh m ed iu m , containing 100 ng/ml PMA in the presence or absence of 0.45 M sucrose at pH 5.7 for 1 h at 37°C. The cells were cooled by washing 3 x with BM, and the amount o f CD4 remaining at the cell surface detected using 0.3 nM ^^^I-Q4120 for 2 h at 4°C with shaking. Unbound ligand was removed with 4 changes of BM and 2 changes of PBS at 4°C, and the cells were harvested in 400 p i of 0.2 M NaOH. The wells were rinsed with 400 pi of H2O which was p ooled with the respective lysate and the sam ples were counted in the gamma counter.

2.7 Cell Surface CD4 Endocytosis Assay.

H eLa-C D 4 cells in 16 mm d iam eter tissue culture wells were cooled on ice for 10 min before briefly washing twice with BM at 4°C. A saturating concentration ( 8 nM) of i25I_q4120 was added to the cells and they were incubated at 4°C for 2 h with gentle shaking. Unbound ligand was removed using 3 quick washes with cold BM and duplicate aliquots of cells warmed to 37°C in BM in the presence or absence of 100 ng/ml PM A for times up to 120 min. Cells were returned to ice and cooled rapidly by washing 3 times with cold BM. To determine the proportion of internalized ligand, one of the duplicate cell aliquots was treated with elution m edium (RPMI 1640 lacking bicarbonate, supplemented with 0.2% BSA, and 10 mM MBS, pH 2.00). The cells were washed twice in cold elution m edium then incubated in cold elution m edium for 2 X 3 min at 4°C. The other aliquot of cells was harvested directly. Cells were harvested in 400 p i 0.2 M NaOH and the wells rinsed with 400 p i H2O which was then pooled with the respective cell lysate. The lysates were counted in the gamm a counter, and the

proportion of acid resistant to total cell counts was calculated for each time point and plotted.