Equal amounts of exponentially growing cells (9 × 106 to 1 × 107cells) were added to 20% trichloroacetic acid, harvested by centrifugation at 3000 rpm for 1 min, and then snap-frozen in liquid nitrogen. Protein was extracted as described previously (144) but without phosphatase treatment. Proteins were resuspended in 1% SDS, 1 mM EDTA, and 100 mM tris-HCl (pH 8.0) containing 10 mM N-ethymaleimide. Protein concentrations were estimated using the bicinchoninic acid protein assay (Thermo Fisher Scientific), and equal amounts of protein (5 to 10 μg) were resolved on 8% SDS-PAGE gels, followed by transfer to nitrocellulose membrane and analysis by immunoblotting as previously described (145) using the following antibodies: anti-myc (9E10, Santa Cruz Biotechnology), anti-tubulin (anti-Tat1), phospho-PKA substrate antibody (RRXS*/T*, 100G7E, Cell Signaling Technology), and anti-Pka (V5 epitope monoclonal antibody, V8012, Sigma-Aldrich). Images were collected using enhanced chemiluminescence reagent (Pierce ECL Plus, Thermo Fisher Scientific) and ImageQuant software (Typhoon FLA9500) or VILBER Fx6 / Fx7 Chemiluminescent System (Labtech).