Biochemical characterization of purified protein. A.

<p>Capillary electrophoresis of purified fraction D was performed using amine capillary column 57 cm long as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057919#s2" target="_blank">Materials and methods</a>’. The amount of protein added to the column was 1 µg. The result showed only a single peak at 2.14 min, indicating the presence of a protein of a single molecular weight. <b>B.</b> Coomassie and silver stain of H1 eluate was performed upon resolving the protein in 8% SDS-PAGE according to the procedure mentioned in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057919#s2" target="_blank">Materials and methods</a>’. <b>C</b>. Dot-blot of purified fraction D using the polyclonal antibody against rabbit<b>.</b> 1, 2, 3, 4, 5, and 6 indicated BSA, 20, 10, 5, 2, and 1 µg of purified protein, respectively. <b>D</b>. Induction of cytochrome P4501A1 in carbofuran-treated hepatocytes. Cultured catfish hepatocytes were treated with different doses of carbofuran for 24 h. Lysate was made, and protein was run on 8% SDS-PAGE gel. Immunoblot was performed as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057919#s2" target="_blank">Materials and methods</a>’ using the antibody against purified protein. Lane1, marker; lane 2, control; lanes 3–7, microsomal protein obtained from hepatocytes treated with various doses of CF: 0.1, 1.0, 10, 100, and 1000 nM, respectively. <b>E.</b> Immunoblot was performed by running all the fractions in 8% SDS-PAGE. The antibody raised against fraction D was used as a primary antibody. The rest of the procedure was followed as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057919#s2" target="_blank">Materials and methods</a>’.</p

Analysis of variance (ANOVA) of CD data using the statistical software SAS (Cary, NC).

<p>Each attempt was replicated at least three times, and values of three observations for each point were considered for statistical analysis.</p>*<p>indicated the values are significant at p<0.05;</p>**<p>indicated the values are significant at p<0.01 level; NS, not significant.</p

SDS-PAGE electrophoresis and chromatogram of eluted protein. A.

<p>SDS-PAGE (8%) was performed of polyethylene glycol (PEG) fractionated protein from solubilized microsome. Lane1, marker; lane 2, microsome; lane 3, solubilized microsome with CHAPS and sodium-cholate; lanes 4–6; solubilized microsomal protein with 10, 20, and 30% PEG, respectively. <b>B.</b> Chromatogram of eluted protein from DE-52 column. Protein was eluted from the column by linear gradient of KCl, and 10-ml fractions were collected. Four conspicuous peaks in terms of protein content measured at 280 nm were observed in the eluate as; Hb, A, B, C, and D (upper graph). Fractions were pooled, and heme content was tested at 416 nm (lower graph). <b>C.</b> The pooled fractions of D peak were added to the equilibrated hydroxyapatite column. The protein was eluted from the column by step gradient of phosphate buffer, and 5-ml fractions were collected. Fractions were pooled. There were two peaks, H1 and H2, in terms of protein content (upper graph). The lower graph represents the heme content of the pooled fractions.</p

Treatment with carbofuran increased phospholipid in catfish livers.

<p>Injection of carbofuran and extraction of liver from catfish were performed according to ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057919#s2" target="_blank">Materials and methods</a>’. Total lipid was isolated from membrane fraction of liver, and phospholipid was estimated from total lipid. * indicates that the value is statistically significant, at <i>p</i><0.05.</p

EROD activity of various fractions in reconstituted system.

<p>EROD activity was reconstituted from fractions of various column eluates described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057919#s2" target="_blank">Materials and methods</a>’. Each value represents the mean ± SE from at least three observations. ND represents ‘Not Detected’.</p

Role of mTOR in CXCL12-induced signaling and cell migration.

<p>(A) Whole cell lysates from CEM stable clones carrying either empty vector (EV) or shRNA constructs (sh-222 and sh-224) were analyzed by western blot analysis to determine the levels of total p70^S6K1. (B) Whole cell lysates from EV and sh-222 clones treated with CXCL12 for 30 minutes were analyzed by western blot analysis to determine the levels of phospho-p70^S6K1. (C) CEM cells were pretreated with rapamycin (100 ng/ml) and KU-0063794 (1 µM) for 1 hour, and then CXCL12 treatment was done for 30 minutes. Equal amounts of whole cell lysates were analyzed by western blot analysis. (D) Migration assay for EV, sh-222 and sh-224 clones were performed similarly as primary T cells described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024667#pone-0024667-g001" target="_blank">Fig. 1A</a>. (E) Surface expression of CXCR4 for EV, sh-222, and sh-224 were determined by FACS analysis, and the levels are expressed as mean fluorescence intensity (MFI). (F) Confocal microscopy to monitor CXCL12-induced actin polymerization in EV, sh-222, and sh-224 clones were performed similarly as primary T cells described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024667#pone-0024667-g001" target="_blank">Fig. 1B</a>. (G) In vivo migration of EV and sh-222 clone mediated by CXCL12 was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024667#s3" target="_blank">Materials and Methods</a>. Tissue staining from individual mice is shown here.</p

Rapamycin blocks CXCL12 induced migration and actin polymerization of T cells.

<p>(A) Primary human T cells were labeled with calcein (5 µM) for 1 h in media and washed. Pretreatment of labeled cells was done with or without rapamaycin (100 ng/ml) for 1 h. Cells (1×10⁵ in 100 µl) were placed in the upper wells of 24-well transwell migration chambers with 5 µm pores (Corning, Corning, NY). In the lower wells, either medium alone or CXCL12 (100 ng/ml) was added to a total volume of 600 µl, and the chambers were incubated for 2 hours at 37°C in 5% CO2 incubator. Triplicate well determinations were performed for each treatment. The level of fluorescence of cells migrating across the chamber was assessed using a microfluorimeter. * indicates the value is statistically significant at p<0.05 level (n = 4). PMI indicates ‘percent migration index’. (B) Primary human T cells were treated with CXCL12 for 30 minutes in the presence or absence of pretreated rapamycin. Confocal microscopy was done according to the procedure mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024667#s3" target="_blank">Materials and Methods</a> to monitor actin polymerization. Red color indicates the actin polymerization. Representative images are shown from each treatment group. (C) Effect of rapamycin and KU-0063794 (KU) on CXCL12-induced cell migration, and effect of MIP3β and TARC in the presence or absence of rapamycin on the migration of resting T cells were performed as described in panel A. (D) Migration assay for CEM cells was performed similarly as primary T cells described above. (E) Dose response curve for rapamycin effect.</p