List of identified differentially expressed proteins in Cry1Ab treated IPEC-J2 cells.

a)<p>Theoretical isoelectric point (pI).</p>b)<p>Accession number in NCBI database.</p>c)<p>Average ratio (Cry1Ab treated/non-treated control) indicates the value derived from the normalized spot volume standardized against the intra-gel standard provided by DeCyder software analysis.</p>d)<p>Peptide coverage, the amount of the identified proteins that the peptides covered.</p

Effect of Cry1Ab on viability of IPEC-J2 cells.

<p>IPEC-J2 cells were incubated with indicated concentrations of Cry1Ab for 24 h and 48 h, respectively. Data are from one representative experiment out of 2 performed. (A) The Cell Proliferation Reagent WST-1 (Roche) was used to measure mitochondrial dehydrogenase activity of viable cells. Valinomycin was used as cell damaging control. Bars represent the mean +/−S.D. of 5 replicate wells. (B) The CellTiter-Glo Luminescent Cell Viability Assay (Promega) was used to estimate the number of viable metabolically active cells based on quantitation of ATP. Bars represent the mean +/−S.D. of 5 replicate wells. (C) The CytoTox-ONE™ Homogenous Membrane Integrity Assay (Promega) was used for estimating the number of dead cells by release of LDH from damaged cells. Bars represent the mean +/−S.D. of 12 replicate wells.</p

Proteomic profiling of IPEC-J2 cells in response to Cry1Ab treatment as revealed by 2D DIGE analysis.

<p>Image shows one representative spot map of Cry1Ab treated IPEC-J2 cell extracts (n = 5) indicating spot boundaries of proteins, whose expression level is increased (red) or decreased (green) in comparison with the corresponding untreated controls (only medium) (P<0.05) as revealed by Decyder V.7.0 software. Spots marked with a number, correlating to the identified proteins in Table1.</p

Increase of Hsp70 protein expression in Cry1Ab-treated IPEC-J2 cells.

<p>IPEC-J2 cells were treated with Cry1Ab for 24 hours and the Hsp70 protein was determined by ELISA and Western blot (insert). For ELISA Hsp70 is normalized to µg protein extract used in the assay. The normalized data are represented as mean relative to the control. Data represent the mean +/− SD; n = 5 different experiments; (*) results are significantly different as compared with untreated controls (P<0.05). For Western blotting the membranes were additionally incubated with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mouse monoclonal antibody (Abcam), which serves as control for protein expression.</p

Effect of Cry1Ab on transepithelial electrical resistance (TEER) in porcine intestinal epithelial monolayers.

<p>TEER of IPEC-J2 cell monolayers was measured 24 h and 48 h after application of increasing doses of Cry1Ab (0.1, 0.5, 1 µg/ml) or valinomycin (500 nM) at the apical side [means ± standard deviation; % of initial value t  = 0 h]; n = 3 independent experiments (2–3 cell culture inserts per treatment group per experiment).</p