Primary monoclonal anti arpc2 antibody

Phosphoproteomic data were confirmed by Western blot (WB) analysis both, in the training set of patients used for proteomic analysis and in a testing set including 23 different samples (11 chronic antibody-mediated rejection, 6 control transplant recipients, and 6 immunocompetent patients). PBMCs’ phosphoproteins (10 μg) isolated from the training set were separated by one dimensional (1D) SDS gel electrophoresis. After transfer, the membrane was blocked in 5% BSA (PBS-T 0.1%), and then incubated overnight with primary monoclonal anti-ARPC2 antibody (Abcam, Cambridge, UK). Membranes were incubated with the appropriate HRP-conjugated secondary antibody and acquired after the addition of the proper substrate. In the same way, total PBMCs’ proteins (100 μg), isolated from the testing set, were analyzed by WB as described above. After the incubation with primary monoclonal anti-ARPC2 antibody and image acquisition, the membranes were stripped and immunoblotted again with monoclonal anti phospho-Ser antibody (Abcam). Clarity™ Western ECL substrate (Bio-Rad, Hercules, CA, USA) was used to detect protein bands by the Versadoc Molecular ImagerTM (Bio-Rad). Results of densitometry analysis were expressed in arbitrary units. Data were normalized to the total protein content in each sample. The immunoblotting experiments were run at least in duplicate.