Ca2+ Mobilization Induced by Gliadin Figure 1. Effect of p31–43 and p57–68 on [Ca2+]i in Caco-2 cells. (A) and (B) Single-cell traces representative of the effect of 20 mg/ml p31–43 and p57–68, respectively, on [Ca2+]i. Starting time of perfusion is indicated by the arrows. Ionomycin (1mM) was added as control (grey arrows) at the end of the experiment. (C) and (D) Dose-dependent effect of p31–43 and p57–68, respectively, on [Ca2+]i increase. PCTR (20 mg/ml) served as control. From 40–65 cells were monitored in each experiment. Each bar represents the mean 6 SEM of data obtained in three independent experimental sessions. *p,0.05 versus its respective control (basal values); **p,0.05 versus previous concentrations and control. doi:10.1371/journal.pone.0045209.g001 according to the manufacturer’s instructions. The first-strand cDNA synthesis reaction was performed using the Maxima first- strand cDNA synthesis kit (Fermentas, Milan, Italy) and 1 mg total RNA. The cDNA was then used to amplify human tTG, GRP78, and CHOP transcripts with the following primers: tTG upper, TGCTGTGGAGGAGGGTGACT; tTG lower, AC- CAGGCGTTGAAGAGCAAA; GRP78-upper, 59-CTGGGTA- CATTTGATCTGACTGG-39, GRP78-lower, 59- GCATCCTGGTGGCTTTCCAGCCATTC-39; CHOP-upper 59-CTT GGC TGACTG AGG AGG AG-39, CHOP-lower, 59- TCA CCA TTC GGT CAATCA GA-39. The concentration of mRNA was normalized to the concentration of GAPDH, which was obtained with the following primers: upper, 59-TTCAA- CAGCGACACCCACTG-39; lower, 59- CACCCTGTTGCTG- TAGCCA-39. For PCR reactions cDNA samples were analyzed in triplicate with the iQTM SYBR Green Supermix (Bio-Rad Laboratories, Milan, Italy) and using the iQTM 5 Multicolor Real Time PCR Detection System (Bio-Rad Laboratories). PCR reactions were performed with 250 nM of each primer and 10 ml of SYBR Green Supermix, in a total volume of 20 ml. The PCR program started with 3 min of incubation at 95uC, followed by 40 cycles of 15 sec at 95uC, 15 sec at 60uC, and 20 sec at 72uC. Western Blot Analyses Caco-2 cells were incubated for 24 or 48 h with 20 mg/ml of p31–43 or p57–68 or, alternatively, with 1 mM THP. Cells were washed with phosphate-buffered saline solution and mechanically harvested in lysis buffer, consisting of 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM dithiotreitol, 0.1% sodium dodecyl sulphate, 1% triton X-100, 1 mM ortovanadate, and inhibitors cocktail (Sigma-Aldrich). After 30 min of incubation on ice, cell extracts were centrifuged at 12,000 g for 30 min at 4uC, to remove cell debris, then 75 mg of total proteins were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to a membrane. To detect tTG, the blot was treated as described elsewhere [23]. To detect GRP78 and CHOP, the blots were treated with 5% skim milk in phosphate- buffered saline overnight at 4uC, then incubated for 2 h with an anti-GRP78 rabbit polyclonal antibody (H-129) (1:500; Santa Cruz, CA, USA), or with an anti-CHOP mouse monoclonal antibody (1:1,500; Santa Cruz), respectively, in phosphate- buffered saline containing 0.1% tween-20. After washing, the blots were incubated for 1 h at room temperature with an anti- rabbit-peroxidase secondary antibody (1:10,000; Bio-Rad Labo- ratories), or with an anti-mouse-peroxidase secondary antibody (1:10,000; Bio-Rad Laboratories), respectively. Immunocomplexes PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e45209