Western Blot Analysis of Cellular Signaling Proteins

Protein was extracted, quantified, and probed with antibody, as previously described.^{69 (link)} Whole cell lysates were extracted using RIPA buffer (Cell Signaling). Nuclear and cytoplasmic lysates were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Kit, according to the manufacturer (Thermo Scientific). Antibodies included AKT (Cell Signaling, Danvers, MA), phospho-AKT (S473) (Cell Signaling), CXCR4 (Abcam, Cambridge, MA), phospho-CXCR4 (S339) (Abcam), eEF2 (Cell Signaling), GRK4-6 (EMD Millipore, Billerica, MA), GRK5 (C-20) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), KCNA1 (Abcam), Lamin B1 (Cell Signaling), NPR3 (Abcam), WIP1 (Bethyl, Montgomery, TX), and β-actin (Sigma-Aldrich, St. Louis, MO). Secondary antibodies Alexa Fluor 680 goat anti-mouse IgG (Life Technologies) or IRDye 800 goat anti-rabbit IgG (Rockland, Gilbertsville, PA) were used at a dilution of 1:5,000. Immunoblots were imaged and quantified, as previously described.^{27 (link)}