Anti lcb

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)].

The cells were lysed with 1% NP-40 in a solution of 0.05 M Tris–HCl (pH 7.5), 0.15 M NaCl, 0.01 M MgCl2, and a protease inhibitor cocktail (P8340; Sigma-Aldrich). After centrifuging the samples at 16,600 × g for 15 min, the supernatant was collected and its protein content was quantified by BCA protein assay (Thermo Fisher Scientific). More than 20 µg of total protein was electrophoretically separated in a SDS–polyacrylamide gel and transferred onto polyvinylidene fluoride membranes, which were incubated for 24 h at 4 °C with each of the following antibodies (1:4,000 dilution): anti-GAPDH, anti-LCB, anti-AMPK, anti-p-AMPK, anti-β-actin, anti-p-ULK1 (Ser555), anti-phosphoULK1 (Ser757), or anti-ULK1 (D8H5) (Cell Signaling Technology, Danvers, MA, USA). Then, the membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody (GenDEPOT) at a concentration of 1:10,000 for 1 h at 25 °C. The proteins were detected using enhanced chemiluminescence reagent (Thermo Fisher Scientific), and images were captured using an FUSLON SOLOS (Korea Biomics, Seoul, Republic of Korea). The band intensities were analyzed using ImageJ software (NIH, USA). The relative protein levels of LC3B were normalized to GAPDH, phospho-AMPK was normalized to AMPK, and ULK1 phosphorylation was normalized using ULK1.