adjusted to equal volumes and detergent content, and proteins were precipitated with chloroform/methanol (1:4 v/v) for Western blot analyses. Expression and Purification of Recombinant APT-1 The APT-1 cDNA containing a Hisx6 tag was obtained by RT- PCR using specific primers and RNA from HeLa cells. The amplified fragment was cloned into BamHI/EcoRI sites of the bacterial expression vector pRSET-A (Invitrogen, USA). Trans- formed Escherichia coli cells were growth at 37uC in LB medium containing 75 mg/ml ampicilin to an optical density of 0.6. Then, a soluble fraction was generated by sonication, lyzosyme treatment (200 mg/ml, 30 min at room temperature), and centrifugation at 10000 g for 15 min at 4uC. Hisx6-APT-1 was then purified from the soluble lysate using a Ni2+-NTA column (GE Healthcare, Fairfield, VT, USA) according to manufacturer’s instructions. Assessment of N13GAP-43(C3S) In Vitro Depalmitoylation by Purified APT-1 To examine the ability of purified recombinant APT-1 to depalmitoylate N13GAP-43(C3S), S and P fractions were obtained from N13GAP-43(C3S) transfected and nontransfected CHO-K1 cells, respectively, by ultracentrifugation as described above. S0 fractions were cleared and P0 fractions were washed by ultracentrifugation. Membrane binding of soluble N13GAP- 43(C3S) protein was done by incubation of S0 fractions obtained from transfected cells with P0 fractions obtained from untrans- fected cells for 1 h at 4uC. The resultant mixture was then ultracentrifuged for 1 h at 4uC and the pellet fraction (P1) was resuspended in 400 ml APT-1 reaction buffer (50 mM Hepes, 2 mM CaCl2, 0.1 mM EDTA, pH 8.0) containing protease inhibitors (PIM plus 1 mM PMSF) and ultracentrifuged again. The resultant P2 fraction was resuspended in 250 ml APT-1 reaction buffer containing PIM/PMSF and subjected to acyl- protein thioesterase treatment by incubation with recombinant APT-1 as previously described [13,14,25] with some modifica- tions. Reactions were performed by mixing 100 ml of P2 fraction with 2 mg recombinant APT-1 (0.18 mg/ml; APT-1 reactions) or 11.1 ml of APT-1 carrier buffer (1M Tris-HCl:glycerol, 1:1; Control reactions) and incubated at 30uC for 1 h. Then, 300 ml of APT-1 reaction buffer was added to samples and subjected to ultracentrifugation. P fractions were then resuspended in the same reaction buffer. Proteins from resulting fractions were precipitated with 10% trichloroacetic acid as described previously [61] and analyzed by Western blot using an antibody to GFP. As a control, we also evaluated under the same experimental conditions the effect of recombinant APT-1 on membrane binding properties of YFP-K-RasC14 [47]. Confocal Microscopy Confocal images were collected using an Olympus FluoView FV1000 confocal microscope (Olympus Latin America, Miami, FL) equipped with a multi-line Argon laser (458, 488 and 514 nm), two Helium Neon lasers (543 nm and 633 nm, respectively). CFP was detected by using laser excitation at 458 nm; a 458/514 nm excitation dichroic mirror and a 470–500 nm bandpass emission filter. YFP was acquired by using laser excitation at 514 nm, a 458/514 nm excitation dichroic mirror and a 530–570 nm bandpass emission filter. Cherry protein was acquired with a laser excitation at 543 nm, a 488/543/633 nm excitation dichroic mirror, and a 560 nm long pass emission filter. For CFP/YFP or CFP/YFP/Cherry acquisition, images were sequentially acquired in line mode. This minimizes the bleed-through between channels, APT-2-Mediated Deacylation of Peripheral Proteins mainly due to overlapping emission spectra of these fluoro- chromes. i) Live Cell Imaging for Subcellular Distribution Analyses. For subcellular distribution analyses and colocalization of GAP-43 proteins with organelle markers, live cell experiments were performed at 3562uC (Tritech DigiTherm temperature controller and a heating adapter plate, CA, USA) and using a 1006 UplanSApo oil immersion/1.4 (numerical aperture) objective (Olympus, Japan). Images were taken using 3 x digital zoom, and an appropriated pinhole to obtain 1 Airy unit for the fluorochrome of shortest wavelength excitation/emission properties (optical slice 0.8 mm). Images of different cells for each dish were taken in a period no longer than 30 min. Images are representative of at least three independent experiments. ii) Live Cell Imaging for Deacylation Kinetic Measure- ments. For deacylation measurements, cells coexpressing N13GAP-43(C3S)-YFP and N27GalNAcT-CFP, a TGN marker, or N13GAP-43(C3S)-YFP, N27GalNAcT-CFP and Cherry-APT-1 or Cherry-APT-2, were used. Live cell experiments were performed at 20uC on the Olympus FluoView FV1000 confocal microscope to minimize vesicular trafficking of GAP-43 [34,62]. After selection of cells coexpressing all proteins (e.g., N27GalNAc- T-CFP, N13GAP-43(C3S) and cherry-APT-1 or cherry-APT-2), images were acquired in the CFP and YFP channels for deacilation kinetic measurements. A 40 x/1.3 PlanApo objective oil immersion (Olympus) was used with a 5 x digital zoom. The image resolution was 320x320 pixels, the scan speed was 10 ms/pixel and the pinhole adjusted to obtain an optical slice of 3 mm. Single 2D images were taken at a frequency of 1 min-1, for 30 minutes. These acquisition conditions were optimized to acquire the Golgi compartment, to minimize bleaching during acquisition and to sample as fast as possible the amount of N13GAP-43(C3S) at the TGN. Quantitative mage Analysis for Deacylation Experiments All image analysis and quantification procedures were done using ImageJ software. Significant fluorescent pixels were isolated from raw images of each channel (CFP and YFP channels) by applying the following equation: ½Im CBSh,i~½Im CRh,i{3:bsCdh,i½1 Where ½Im CRh,i is the matrix (image) containing the gray values of the raw image of channel i, bCsdh,i is a scalar representing the standard deviation of intensity of background pixels and [1] is a unitary matrix of equal size of ½Im CRh,i. bsd was obtained from a 50650 pixel region from an area empty of cells. Negative elements resulting from the above equation are defined as 0 and consequently the elements of the ½Im CBSh,i matrix are greater or : Ch,iallows discriminate those pixels equal to 0. The threshold 3 bsd whose intensity is significantly different from the background noise. Although the intensity distribution of pixels is not normal, we did not obtain differences by applying the above equation or a threshold containing the 90% of background pixels (data not shown). A mask ½M Ch,i, containing the position of significant pixels in ½Im CBSh,i was generated by applying a threshold value equal to 1, and then substrating a value of 254 in Image J. The resulting image was then converted to 16 bit for compatibility purposes. The ½M Ch,i matrix has values 0 and 1. Then, objects with a diameter smaller than 8 pixels were discarded with the remove objects plugin of Image J. Once obtained½M Ch,i, pixelsij in the raw PLoS ONE | www.plosone.org 12 November 2010 | Volume 5 | Issue 11 | e15045