Tissue arrays were stained with an anti-PGRMC1 antibody raised to a recombinant protein composed of amino acids 43 - 195 of PGRMC1 (ProteinTech Group, Inc., Chicago IL). Immunohistochemistry was performed by the University of Kentucky Histology Laboratory using the Dako Envision kit (Carpinteria, CA) and following the manufacturer's instructions. Staining was analyzed and scored by a pathologist (Dr. Stewart), as well as other authors, and analyzed statistically using Microsoft Excel. Kaplan-Meier curves were analyzed and prepared using Graphpad Prism software. Blocking was performed with an equimolar concentration of purified, recombinant PGRMC1-glutathione S-transferase fusion protein spanning the antigenic region, and the protein has been described [27 (link)].
Protein levels were analyzed by western blot as previously described [27 (link)], blotting electrophoresed proteins to Immobilon-P membranes and developing using the West Pico chemiluminescent substrate (Pierce). Blots were performed at least in duplicate. The antibodies used were the following: anti-GAPDH (Santa Cruz, FL-335), anti-LCB (Cell Signaling, D11), anti-SQSTMI/p62 (Cell Signaling, 5114), anti-caspase 3 (Santa Cruz, sc-7138), anti-PARP (Santa Cruz, sc-7150) and anti-PGRMC1 [4 (link)].
Protein levels were analyzed by western blot as previously described [27 (link)], blotting electrophoresed proteins to Immobilon-P membranes and developing using the West Pico chemiluminescent substrate (Pierce). Blots were performed at least in duplicate. The antibodies used were the following: anti-GAPDH (Santa Cruz, FL-335), anti-LCB (Cell Signaling, D11), anti-SQSTMI/p62 (Cell Signaling, 5114), anti-caspase 3 (Santa Cruz, sc-7138), anti-PARP (Santa Cruz, sc-7150) and anti-PGRMC1 [4 (link)].