Although GRIP1b is palmitoylated in heterologous find more cells, endogenous GRIP1 palmitoylation is not well characterized in neurons. Using [3H]palmitate labeling, we first

confirmed that GRIP1 is indeed palmitoylated in primary neurons (Figure 2E). Immunoblotting of ABE samples with a “pan-GRIP1” antibody also showed a robust signal, confirming GRIP1 palmitoylation in both cultured neurons and in intact brain (Figure 2E). To specifically detect GRIP1b palmitoylation, we probed the same ABE samples with our GRIP1b antibody. Strikingly, this suggested that a higher percentage of GRIP1b is palmitoylated in neurons than the well-known palmitoyl-protein PSD-95 (Figure 3A). Their similar subcellular localization suggests that DHHC5 is appropriately positioned to palmitoylate GRIP1b in neurons. Indeed, although GRIP1b protein was detected as early as 5 days in vitro [DIV] (Figure 2F), GRIP1b palmitoylation was detected only at later times (12–19 DIV), coincident with the appearance of DHHC5 (Figure 2F). Moreover,

neurons infected with lentivirus encoding a small hairpin RNA (shRNA) that specifically targets DHHC5 (Figure S2I) showed markedly Afatinib reduced levels of palmitoylated, but not total, GRIP1 (Figures 2G and 2H). Importantly, as a control for potential off-target effects of shRNA, both DHHC5 levels and GRIP1 palmitoylation were rescued by coexpression of shRNA-resistant DHHC5 (Figures 2G and 2H). DHHC5 knockdown and rescue did not affect either palmitoylated or total levels of the known palmitoyl proteins Fyn (Figure 2G) or SNAP25 (data not shown). DHHC5 knockdown did not completely eliminate GRIP1b palmitoylation, suggesting that other PATs, in particular DHHC8, might compensate for loss of DHHC5. Indeed, while infection of a DHHC8-specific (Figures S2J and S2K) shRNA only slightly reduced GRIP1 palmitoylation, coinfection with shRNAs targeting DHHC5 and DHHC8 together reduced GRIP1 palmitoylation to almost undetectable levels (Figures 2G and 2H). Levels of total and palmitoylated SNAP25 and Fyn were unaffected, even in DHHC5 plus DHHC8 knockdown neurons. These results strongly suggest that both

DHHC5 and DHHC8 can palmitoylate GRIP1 in neurons but that DHHC5 is the major endogenous regulator of GRIP1 palmitoylation. Indeed, an antibody that recognizes both DHHC5 and DHHC8 equally SB-3CT revealed that DHHC5 is by far the major of these two PATs in our neuronal cultures (Figure S2L). Thus, in subsequent experiments we focused our attention on DHHC5. Although palmitoylation is reversible, rates of palmitate turnover on neuronal proteins vary widely (Huang and El-Husseini, 2005 and Kang et al., 2008). Palmitate turnover rate can provide insight into the possible function of palmitoylation; rapid turnover suggests a role in dynamic events such as regulated protein trafficking, while slow palmitate turnover suggests a role in long-term static protein targeting.