Rabbit anti human antibodies

Caco-2 3D cysts in 96-well plates were exposed to plasma from active, inactive CD patients or HC for 24 h at 37 °C and 5% CO₂. Thereafter, cysts were processed for cell-based ELISA and incubated with rabbit anti-human antibodies (Cell Signalling Technology, Leiden, The Netherlands) against p-p38 (#9258P4511P), p38 (#9212P), p-ERK1/2 (#4370), ERK1/2 (#4695), p-JNK (#4668) and JNK (#9252) with 1:100 dilution in the blocking solution Ray Biotech), followed by HRP-conjugated anti-rabbit IgG (Dako, Glostrup, Denmark) as we described previously^{60 (link)}. Finally, 3, 3′, 5, 5′-Tetramethylbenzidine (TMB) was added, followed by stop solution (2N H₂SO₄) and optical density was read at 450 nm by SpectraMax M2 spectrophotometer (Molecular Devices).

Human DPCs cultured in α-MEM were exposed to dental monomers (1mM HEMA, 5mM MMA and 1mM TEGDMA) in the absence or presence of 10 mM NAC for 24 h (n = 3) and lysed in RIPA buffer. Protein contents of the lysed cells were measured with the BCA Protein Assay Kit. Extracted proteins were loaded on 10% sodium dodecyl sulfate polyacrylamide gels, transferred to polyvinylidene fluoride membranes (Bio-Rad), and blocked with 5% nonfat milk powder. Membranes were incubated overnight with the following primary rabbit anti-human antibodies (Cell Signaling Technology, Danvers, MA): anti-Bcl-2, anti-Bax, anti-p53, anti-cleaved caspase-3, and actin. Membranes were incubated with goat anti-rabbit secondary antibodies. Protein signals were visualized by using the ECL Western Blotting Detection System (GE Healthcare, Piscataway, NJ, USA). Protein expression levels were normalized to actin by using Image-Pro Plus 5.0 Software (Media Cybernetics Inc., Bethesda, MD, USA).

Rabbit anti-human antibodies were purchased from Cell Signaling Technology (Danvers, MA) to probe the expression of caspases (caspase-8, caspase-9, cleaved-caspase-9 and caspase-3, cleaved-caspase-3), Bcl-2 family members (Bcl-2, Bcl-xl, Mcl-1 and Bad) and the sub MAPK family (p38MAPK and p-p38MAPK) by Western blot. Rabbit anti-human antibodies manufactured by Epitomics (Burlingame, CA) were selected to probe the protein expression levels of cytochrome C, p-Bad and Bax by Western blot. ProteinTech Group (Chicago, IL)-manufactured rabbit anti-human JNK, p-JNK, ERK and p-ERK antibodies were employed to evaluate the expression changes of the corresponding antigens via Western blot. Cationic dye JC-1 was purchased from Biotium (Hayward, CA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Z-DEVD-FMK (caspase-3 inhibitor), Z-VAD-FMK (caspase-9 inhibitor), PD98059 (ERK-specific inhibitor), SB203580 (p38-specific inhibitor), SP600125 (JNK-specific inhibitor) and dimethyl sulphoxide (DMSO) antibodies were purchased from Sigma (St. Louis, MO). An annexin V-FITC apoptosis detection kit was purchased from Pharmingen (San Diego, CA).