Identification of the polyclonal antibody against pFTO.

<p>(A) ELISA of the antiserum against pFTO; (B) Western blot analysis of anti-FTO antibody. Lanes 1 and 2: the purified pFTO protein detected by polyclonal antibody against pFTO.</p

Effect of pFTO on the proliferation of porcine intramuscular preadipocytes.

<p>Porcine intramuscular preadipocytes were seeded in a 24-well plate at a density of 1 × 10⁴ cells/well. When the cells reached about 50% confluence, 0.5 μg of the plasmid pcDNA3.1(+)-pFTO or the empty vector pcDNA3.1(+) was transfected. (A) Cell proliferation was evaluated by EdU proliferation assay after 24 h of transfection. The Click-it reaction revealed EdU staining (red). The cell nuclei were stained with Hoechst 33342 (blue). (B) The percentage of EdU-positive cells was quantified. Results are presented as mean ± SE (n = 8). ***<i>P</i> < 0.001 as compared with the control group.</p

Subcellular localization of pFTO in porcine intramuscular preadipocytes using an indirect fluorescent immunocytochemical technique.

<p>(A) Endogenous pFTO protein in porcine intramuscular preadipocytes stained first with the anti-pFTO polyclonal antibody and then with FITC-conjugated goat anti-mouse IgG (green). (B) The nucleus marked with DAPI (blue). (C) The merged image.</p

List of genes, primer sequences, GenBank accession numbers, and product sizes in this study.

<p>List of genes, primer sequences, GenBank accession numbers, and product sizes in this study.</p

Tissue distributions of pFTO.

<p>(A) Relative expression levels of <i>pFTO</i> mRNA in different tissues. (B) Western blot analysis of pFTO protein levels in different tissues. The amount of <i>pFTO</i> was normalized to the amount of <i>β-actin</i>. Bars presented the means ± SE (n = 3).</p

Identification of porcine intramuscular preadipocytes.

<p>Porcine intramuscular preadipocytes were characterized by Pref-1 immunofluorescent staining (100×).</p

Expression pattern of <i>pFTO</i> mRNA during porcine intramuscular preadipocytes differentiation.

<p>RNA was extracted from the differentiating porcine intramuscular preadipocytes on the days 0, 3, 5, 7 and 9. <i>pFTO</i> mRNA expression was analyzed by real-time quantitative PCR. Data were the mean and SE from three independent experiments. *<i>P</i> < 0.05, ***<i>P</i> < 0.001.</p

The <i>K_m</i> value of opPPL and commercial PPL against p-NPP determined by Lineweaver-Burk method.

<p>(A) The <i>K_m</i> value of opPPL. (B) The <i>K_m</i> value of commercial PPL.</p

Effect of metal ions on opPPL and commercial PPL activity.

<p>(A),(B),(C) and (D) represent the effect of Zn²⁺, Ca²⁺, Fe³⁺ and Cu²⁺ on the activity of opPPL and commercial PPL, respectively. y means relative enzyme activity; x means concentration of the metal ions; These assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114385#s2" target="_blank">Materials and methods</a> using 10.0 mM p-NPP as substrate(n = 3). The maximum value was taken as 100%.</p

Comparison of opPPL and commercial PPL activity.

<p>(A) Effect of pH on the activity of opPPL and commercial PPL. (B) Effect of temperature on the activity of opPPL and commercial PPL.(C) The thermostability of commercial PPL. (D) The thermostability of purified opPPL. These assays were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114385#s2" target="_blank">Materials and methods</a> using 10.0 mM p-NPP as substrate (n = 3). The maximum value was taken as 100%.</p