Monoclonal mouse igg1 antibody

NHEKs (25,000 cells/well) cultured in white flat-bottom 96-well microplate were co-transfected in the presence of Lipofectamine 2000 (Invitrogen, Grand Island, NY) with a luciferase reporter harbouring an ST18 gene promoter fragment spanning the rs17315309 wild-type (T) or risk (C) allele^{16 (link)}, or with an empty luciferase pGL4.17 as a negative control and a Renilla expression vector. In some experiments, cells were concomitantly transfected with TP53-specific siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, SC-29435) or control siRNA (Life Technologies, Carlsbad, CA, 452002). Efficiency of gene knock down was assessed by qRT-PCR and Western blotting (Supplementary Fig. 8). Twenty four hours post transfection, Ca²⁺ concentration was raised to 1.2 mM and cells were treated with the AK23 monoclonal antibody (3.75 μg/ml) (Biozol, Eching, Germany, D219-3) or a monoclonal mouse IgG1 antibody (3.75 μg/ml) (R&D Systems, Minneapolis, MN,MAB002). Fourty eight hours post transfection, a dual luciferase assay (Promega, Madison, Wisconsin) was used to measure luciferase activity, which was normalized to Renilla luciferase activity, using Tecan Infinite M200 device (Tecan Group Ltd, Männedorf, Switzerland).

Monoclonal mouse IgG1 antibody made against recombinant aSN was purchased from R&D Systems, Minneapolis, MN, USA. Monoclonal rabbit IgG antibody made against aSN epitope 118–123, was purchased from Abcam, Cambridge, MA, USA.

NHEKs were exposed to monoclonal antibody AK23 (3.75 μg/ml) (Biozol, Eching, Germany, D219-3) or monoclonal mouse IgG1 antibody (3.75 μg/ml) (R&D Systems, Minneapolis, MN, MAB002). For IF and Western blotting studies, we used mouse monoclonal anti-p53 antibody (diluted 1:50; Santa Cruz Biotechnology, Santa Cruz, CA, DO-1), mouse monoclonal anti-desmoglein-3 antibody (5H10) conjugated to Alexa Fluor 488 (Novus Biologicals, LLC, NBP1-78984AF488), rabbit polyclonal anti-DDDDK tag (Binds to FLAG tag sequence) antibody (diluted 1:100, Abcam, Cambridge, UK, ab1162), secondary horseradish peroxidase-conjugated goat anti-mouse antibody (diluted 1:10,000; Jackson ImmunoResearch Laboratories, West Grove, PA, 115-035-003), mouse monoclonal anti β-actin antibody (Sigma-Aldrich, diluted 1:10,000, Abcam, Cambridge, UK, ab8224), mouse monoclonal anti α-tubulin antibody (diluted 1:5000, Sigma-Aldrich, T9026), secondary horseradish peroxidase-conjugated anti-rabbit antibody (diluted 1:5000; Sigma-Aldrich, 12-348) and rabbit polyclonal anti p38 MAPK antibody (diluted 1:1000 Cell Signaling Technology, Danvers, MA, USA, #9212).