Mouse monoclonal antibodies against phospho p38

Protein preparation, SDS-PAGE (using 4 –12% gels; Invitrogen), transferred from the gel onto an Immobilon-P^SQ transfer membrane (Millipore Corp, Bedford, MA) were performed at room temperature as previously described [24 (link),52 ]. Antibody labeling was detected by enhanced chemiluminescence (ECL) using primary antibodies: mouse monoclonal antibodies against phospho-p38 (Cell Signaling Technology, Santa Cruz, CA; catalog #: 9216; 1:500 dilution), mouse monoclonal antibodies against p38 (Santa Cruz Biotechnology, Danvers, MA; catalog #: sc-81621; 1:500 dilution) and a mouse monoclonal antibody against GAPDH (Santa Cruz Biotechnology; sc-32233; 1:2500 dilution). Anti-mouse secondary antibodies from Sigma-Aldrich and ECL kit from Pierce (Invitrogen) were used.

Proteins were extracted from HK-2 cells in ice cold PBS in presence of 1% Aprotinin (Sigma Chemicals, St. Louis, MO). Cells were then solubilized in RIPA buffer with protease inhibitors (Thermo-Fisher Scientific, Waltham, MA). Proteins were quantified and loaded into 4-12%, SDS-PAGE and then transferred onto an Immobilon-PSQ transfer membrane (Millipore Corp, Bedford, MA) at room temperature as previously described^{17 (link)}. Antibody labeling was detected by enhanced chemiluminescence (ECL) using primary antibodies including: mouse monoclonal antibodies against phospho-p38 (Cell Signaling Technology, Santa Cruz, CA; 1:500 dilution); mouse monoclonal antibodies against p38 (Santa Cruz Biotechnology, Danvers, MA; 1:500 dilution); rabbit polyclonal antibodies against ERK (Cell Signaling Technology, Santa Cruz, CA; 1:1000 dilution); rabbit polyclonal antibodies against phospho-ERK (Cell Signaling Technology, Santa Cruz, CA; 1:1000 dilution).