Polyclonal goat anti rabbit hrp

Freshly isolated monocytes were cultured in the presence or absence of TsSP (40 μg/ml) for 30 min. Cell lysates were prepared using NP-40 lysis buffer (1 % Nonidet P-40, 10 % glycerol, 100 mM NaCl₂, 10 mM MgCl₂, 50 mM Tris, pH 7.4) mixed with protease and phosphatase inhibitors (Halt protease and phosphatase inhibitor cocktail, Thermo Fisher Scientific, Rockford, IL, USA). Prior to immunoblotting, lysates were boiled 10 min in Laemmli sample buffer containing 1 % β-mercaptoethanol. Equal sample volumes were subjected to 10 % sodium dodecyl sulfate–PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Membranes were washed with TSM/0,05 % Tween and blocked in TSM/0,05 % Tween/10 % roti-block (Techmate, Milton Keynes, UK, #A151.4). The following primary antibodies (in TSM/0,05 % Tween/5 % BSA) were used: PhosphoPKC (Cell signaling, Beverly, MA, USA, #9371S) and GAPDH (Santa Cruz Biotechnology, Heidelberg, Germany, #sc-32233) as a loading control. Followed by incubation with polyclonal goat anti-rabbit HRP (Dako, Heverlee, Belgium, #p0448) and polyclonal rabbit anti-mouse HRP (Dako, Heverlee, Belgium, #p0161). Proteins were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, USA), in an EpiChemi II Darkroom (UPV Laboratory Products). Bands were quantified using Image J.