The GFP fill was then used to trace 20 μm segments along the three most prominent dendrites emanating from the cell body. Each dendritic segment was then outlined in the GRIP1-myc channel and the average fluorescence intensity logged to a spreadsheet. The analysis was repeated at 20 μm steps for each image. Similar analysis was performed to determine the dendritic distribution of transfected DHHC5 (wild-type and mutants) and DHHC3. To quantitate dendritic puncta of transfected GRIP1, each dendritic segment (outlined in the GRIP1-myc channel as above) was thresholded by gray value at a level close to 50% of the dynamic range. This threshold value

was kept constant for all images this website in each condition, and background noise from these images was negligible. The same dendritic regions were outlined as above, the software was used to count puncta of 2–20 pixel units within each segment, and the results were logged to a spreadsheet. The analysis was repeated at 20 μm steps for each image. To analyze the effect of 2-Bromopalmitate on endogenous GRIP1 puncta, images were initially thresholded as above. A new image was generated of those puncta that overlapped a GFP-transfected neuron within the same field. This allowed GRIP1 signals to be assigned to dendrites emanating from a specific neuron with a defined center. The soma was traced and masked manually, and the GFP signal was used to trace 20 μm dendritic segments as above. Puncta in

each dendritic segment were Sirolimus chemical structure counted as above and logged to a spreadsheet. Live imaging was performed essentially as described (Lin and Huganir,

2007 and Thomas et al., 2008). Briefly, tuclazepam neurons on coverslips were transfected with pH-GluA2 plus vector, GRIP1b wild-type or mutants, or HA-DHHC5. Seventy-two hours after transfection, coverslips were assembled in a chamber perfused with imaging buffer (Lin and Huganir, 2007 and Thomas et al., 2008). A single neuron was selected based on pHluorin signal, and baseline fluorescence was monitored for 10 min prior to perfusion for 5 min with 20 μM NMDA in low-magnesium imaging buffer (Lin and Huganir, 2007 and Thomas et al., 2008) and subsequent recovery in standard imaging buffer. Using ImageJ, the change of pHGluA2 fluorescence in both the cell soma and in a single primary dendrite was monitored and logged to a spreadsheet. We gratefully acknowledge Drs. Masaki and Yuko Fukata (National Institutes of Natural Sciences, Okazaki, Japan) for mouse DHHC cDNAs, and Dr. Y. Igarashi (Hokkaido University, Japan) for human DHHC5 and DHHC8 cDNA. We thank Mrs. Min Dai for neuronal “Banker” cultures, Mr. R. Johnson for the myc-FUW vector and KBD construct, Mrs. L. Hamm for expert technical assistance, Dr. C.-Y. Su (Yale University) for comments on the manuscript, and Dr. V. Anggono and all other R.L.H. lab members for helpful discussions. This work was supported by funding from the Howard Hughes Medical Institute and the NIH (R01 MH64856).