APT-2-Mediated Deacylation of Peripheral Proteins Figure 3. GAP-43 deacylation is not mediated by APT-1. A) PCR analysis of APT-1 expression using specific primers and the following templates: the reaction mixture [ctrl (-)]; a plasmid containing the cDNA coding APT-1 [ctrl (+)]; genomic DNA from CHO-K1 cells (gDNA CHO-K1); first- strand cDNA obtained from RT of CHO-K1 cells purified mRNA (RT-PCR CHO-K1 cells); genomic DNA from HeLa cells (gDNA HeLa) and first-strand cDNA obtained from RT of HeLa cells purified mRNA (RT-PCR HeLa cells). Genomic DNA and mRNA were extracted from CHO-K1 and HeLa cells. B) PCR analysis of APT-1 expression using as template: first-strand cDNA obtained from RT of HeLa cells purified mRNA [ctrl (+)]; the reaction mixture [ctrl (-)] and first-strand cDNA obtained from mRNA of hamster brain (RT-PCR Brain), liver (RT-PCR Liver) and ovary (RT-PCR Ovary). C) 60 h after transient transfection, CHO-K1 cells expressing N13GAP-43(C3S) alone (Control, upper panels) or coexpressing N13GAP-43(C3S) and APT-1(APT-1, lower panels) were treated with 50 mM 2-BP. CHX and protein degradation inhibitors were added 30 min before imaging and maintained in the culture media until the end of each experiment. GAP-43 subcellular distribution was monitored by live cell confocal microscopy. Each panel shows image from YPF fluorescence (pseudocoloured gray) at 0, 5, 10 and 15 min from 2-BP addition. The insets in each panel show (left to right): YFP fluorescence (pseudocoloured green), CFP fluorescence (pseudocoloured red) and the merge of both fluorescence signals. D) Quantification [N13GAP-43(C3S) associated to TGN membranes] of images showed in panels C (see Materials and Methods). E) 60 h after transient transfection, CHO-K1 cells expressing H-RasG12V (upper panels) or coexpressing H-RasG12V and APT-1 (lower panels) were treated with 50 mM 2-BP (2-BP) or vehicle (Ctrl) in presence of CHX and protein degradation inhibitors for 0, 3 and 6 h and the subcellular distribution analyzed by live cell confocal microscopy. Each panel shows image from YFP fluorescence (pseudocoloured gray). The insets show the cells expressing APT-1 (cherry fluorescence). F) S fraction from PLoS ONE | www.plosone.org 6 November 2010 | Volume 5 | Issue 11 | e15045