A Neural Correlate of Working Memory in the Monkey Primary Visual Cortex

APP. For Tip60, both a partial rat cDNA and fulllength human cDNA were analyzed (22). 29. J. Kamine, B. Elangovan, T. Subramanian, D. Coleman, G. Chinnadurai, Virology 216, 357 (1996). 30. Q. Ran, O. M. Pereira-Smith, Gene 258, 141 (2000). 31. T. Yamamoto, M. Horikoshi, J. Biol. Chem. 272, 30595 (1997). 32. T. Ikura et al., Cell 102, 463 (2000). 33. The GST pull-down assays were performed essentially as described (46), by using purified wild-type and mutant rat GST-Tip60b, APP, and Myc-Fe65 expressed by transfection in COS cells. Extracts from transfected COS cells were incubated for 4 hr at 4°C with 10 mg of GST-Tip60 or GST-Tip60* bound to glutathione agarose. Beads were washed five times in 0.15 M NaCl, 50 mM Hepes-NaOH, pH 7.5, 1% IGEPAL CA-630 (Sigma) resuspended in 100 ml SDSpolyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, and 20 ml were analyzed by SDS-PAGE and immunoblotting using antibodies to APP and the HAand Myc-epitopes. 34. C. Zwahlen, S. C. Li, L. E. Kay, T. Pawson, J. D. Forman-Kay, EMBO J. 19, 1505 (2000). 35. C. Dalluin et al., Mol. Cell 6, 921 (2000). 36. Y. Yan, N. A. Barlev, R. H. Haley, S. L. Berger, R. Marmorstein, Mol. Cell. 6, 1195 (2000). 37. HeLa cells plated on cover glass in a 12-well plate were transfected with pcDNA3.1-N-HA-Fe65, pCMVMyc-hTip60, and pCMV5-APP (0.2 mg each) using Fugene6 (Roche). Two days after transfection, cells were washed two times with PBS, fixed (3.7% formaldehyde for 10 min at room temperature), and blocked and permeabilized in PBS containing 3% bovine serum albumin, 0.1% IGEPAL CA-630 for 20 min. Cells were then incubated with mouse monoclonal antibody against HA (BabCo, Berkeley, CA), rabbit polyclonal antibody against APP, and goat polyclonal antibody against Myc (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 hour, washed three times with PBS, and treated with the appropriate Cy2-, Cy3-, and Cy5-labeled secondary antibodies ( Jackson Laboratories) for 1 hour. After three washes with PBS and one wash with water, cells were mounted and observed by microscopy. 38. G. Minopoli et al., J. Biol. Chem. 276, 6545 (2001). 39. For transactivation assays of Gal4-Tip60, COS, HeLa, and HEK293 cells were cotransfected with one of the following: (i) pG5E1B-luc (Gal4 reporter plasmid); (ii) pCMV-LacZ (b-galactosidase control plasmid); (iii) pMst (Gal4); pM-hTip60 (full-length wild-type human Gal4-Tip60b); or pM-hTip60* (full-length mutant human Gal4-Tip60b); (iv) pCMV5-Fe65 (Fe65), pCMVMyc-Fe65(242-711) [Fe65(242-711)], pCMV5Fe65(1-553) (Fe65D PTB2), or pCMV5-Fe65mW4 (Fe65mW4) where indicated; and (v) pCMV5-APP (human APP695) or pCMV5-APP* (mutant human APP695). All transfections contained one of the plasmids listed in (i to iii), whereas (iv) and (v) were variable. Analyses were performed as described in (19). 40. M. S. Brown, J. Ye, R. B. Rawson, J. L. Goldstein, Cell 100, 391 (2000). 41. Y. M. Chan, Y. N. Jan, Cell 94, 423 (1998). 42. B. L. Sopher et al., Brain Res. Mol. Brain Res. 26, 207 (1994). 43. D. C. Lu et al., Nature Med. 6, 397 (2000). 44. A. B. Vojtek, S. M. Hollenberg, J. A. Cooper, Cell 74, 205 (1993). 45. M. Okamoto, T. C. Südhof, J. Biol. Chem. 272, 31459 (1997). 46. Y. Hata, C. A. Slaughter, T. C. Südhof, Nature 366, 347 (1993). 47. X. Cao, T. Südhof, unpublished observations. 48. We thank C. Haass (Munich, Germany), N. Huang and W. Miller (UCSF), R. Roeder (Rockefeller University), J. Herz, E. Olson, J. Lu, M. Lush (University of Texas, Southwestern), K. Beyreuther (Heidelberg, Germany), and R. Jahn (Goettingen, Germany) for reagents and advice. We are grateful to S. Sisodia, M. S. Brown, J. L. Goldstein, E. H. Koo, and D. Selkoe for advice and discussions.