image with significant intensity values were selected by ½Im CSPh,i ij ~ ½Im RCh,i ij : ½M Ch,i ij i.e, by multiplying raw and mask images using the image calculator tool. For TGN masks (i.e. those derived from N27GalNAcT-CFP images) the background level in the CFP channel was determined around the organelle and not from an area empty of cells since in our conditions a fraction of N27GalNAc-T-CFP was present in the endoplasmic reticulum. After creating each TGN mask, we measured the area of TGN in pixel number units as: AreaTGN ~sum ½M TGN ROI Where TGN refers to the CFP channel. The sum of the elements of the mask was performed over a ROI that completely involves the organelle in all the time series of a single cell. By multiplying each ij pixel of the ½Im SYPFP(the 2-D matrix containing the significant N13GAP-43(C3S)-YFP data) by the corresponding of ½M TGN , we obtain ½Im TYGFPN ij ~ ½Im SYPFP ij : ½M TGN ij and the integrated YFP fluorescence intensity inside the organelle is obtained by FluorescenceTYGFPN ~sum ½Im TYGFPN ROI Where the ROI is the same that was employed to determine TGN area. The N13GAP-43(C3S)-YFP concentration is determined by dividing FluorescenceTYGFPN by AreaTGN . N13GAP{43ðC3SÞ{YFP TGN ~ FluorescenceTYGFPN AreaTGN Because we were interested how GAP-43 is distributed between TGN and cytoplasm and the above parameters scaling up with protein expression, we measured the TGN to cytoplasm concentration ratio at each time i as follow: Kp N13GAP{43ðC3SÞ{YFP TGNðiÞ ~ N13GAP{43ðC3SÞ{YFP TGNðiÞ ½N13GAP{43ðC3SÞ{YFP CytðiÞ Where TGN and Cyt refers to the TGN and cytoplasm, respectively. The concentration of GAP-43 at the cytoplasm was determined by measuring the average gray value (integrated gray value/number of pixels of the ROI) of GAP-43 in a small region of interest located in the cytoplasm. Typically, the cytoplasmic ROI area was comparable to that of TGN marker area. Thus, data acquisitions are independent of organelle size and initial steady APT-2-Mediated Deacylation of Peripheral Proteins state conditions. Moreover, these parameters were also indepen- dent of GAP-43 expression levels and TGN size, as previously described [34]. For GAP-43 deacylation, we considered that after an infinite period of acylation inhibition in the presence of an active deacylation mechanism, the amount of TGN associated GAP-43 completely vanish. At this point, GAP-43 is completely dissociated from TGN and only localizes at the cytosol as a soluble protein. For this reason, we expect that the Kp parameter reach 1 at this time. In this sense, it was considered more appropriate to describe the acylation state measured as the amount of TGN-membrane associated GAP-43 as follow: TGN{membrane:associatedGAP{43~ N13GAP{43ðC3SÞ{YFP TGNðiÞ Kp Kp ½N13GAP{43ðC3SÞ{YFP TGNð0Þ {1 N13GAP{43ðC3SÞ{YFP TGNðiÞ Where Kp and Kp N13GAP{43ðC3SÞ{YFP TGNð0Þ are the TGN to cytoplasm concentration ratios of N13GAP-43(C3S) at time i and 0, respectively. mRNA Purification and RT-PCR Animal handling was performed according to the standards stated in the Guide to the Care and Use of Experimental Animals published by the Canadian Council on Animal Care and approved by the local Institution (Facultad de Ciencias Qu´ımicas, Uni- versidad Nacional de Co´ rdoba, Argentina, Exp. Nu 15-99-40426). For hamster tissue mRNA purification and analyses, hamsters were anesthetized and different organs extracted. Then, the tissues were homogenized with an Ultraturrax tissue disintegrator and lysates were passed through a Dounce homogenizer until homogeneity. Then, the cleared lysates were centrifuged at 13000 g and mRNA or genomic DNA were extracted using mRNA and genomic DNA extraction kits from GE healthcare and Sigma-Aldrich, respectively. For tissue cultured cells, 24 h after seeding or differentiation, cells were washed with cold phosphate buffered saline (PBS) and harvested by scrapping in PBS. Extracts were centrifugated at 13000 g for 15 min and mRNA or genomic DNA purified using the commercial extraction kits, according to manufacture instructions. After purification, reverse transcriptase-PCR was performed using 2 mg of purified mRNA as template. Alternative- ly, PCR was performed using 50 ng of genomic DNA. Primers are described in supplemental Table S1. Bioinformatic Search for Acyl-Protein Thiosterases First, we performed a Blast search for nonredundant human protein sequences (nonredundant human database) which align to PPT-1 (NP_000301.1) and/or APT-1 (NP_006321.1) protein sequences [63,64]. In addition, we attempted to identify members of PPT-1 and/or APT-1 families in Pfam databases (http://pfam. sanger.ac.uk/) [#PF02230 (APT-1) and #PF02089 (PPT-1)] [65]. Overall, we obtained four groups of proteins. After discarding redundant sequences, the resulting proteins were grouped according to their coding genes. Finally, we obtained five different genes coding for 21 mRNA transcripts which are potentially involved in the expression of acyl-protein thioesterase enzymes (Table S1). PLoS ONE | www.plosone.org 13 November 2010 | Volume 5 | Issue 11 | e15045