Phospho pka c thr197

BRIN‐BD11 cells were seeded into 100‐mm tissue culture dishes at a density of 2 × 10⁶ cells/dish for 24 hours prior to 6‐hours serum‐free starvation, Phylloseptin‐PBu (10⁻⁷ mol/L) was then added for 16‐hours incubation. The cells were rinsed twice with ice‐cold PBS, solubilized in lysis buffer containing 50 mmol/L Tris (pH 7.5), 1% Nonidet P‐40, 150 mmol/L NaCl, 2 mmol/L EGTA, 1 mmol/L Na₃VO₄, 100 mmol/L NaF, 10 mM Na₄P₂O₇, 1 mmol/L phenylmethylsulfonyl fluoride, 10 μg/mL aprotinin and 10 μg/mL leupeptin. After centrifugation at 14 000× g for 10 minutes at 4°C, the supernatant was boiled for 5 minutes in SDS‐PAGE sample buffer [50 mmol/L Tris‐Cl (pH 6.8), 2% sodium dodecyl sulphate, 10% glycerol, 2% β‐mercaptoethanol and 0.004% bromophenol blue] and separated by 7.5% SDS‐PAGE, and transferred to nitrocellulose membrane (GE Healthcare Life sciences, UK), and detected by Western blotting with the indicated antibody (Cell Signaling Technology, UK) using enhanced chemiluminescence [Pierce^® ECL Western Blotting Substrate (Thermo scientific, USA)], and imaged via Chemi Doc™ MP system (Bio‐Rad, UK).
The antibodies that chosen for the present study were PKA‐Cα (1:1000; Cell Signaling, #4782), phospho‐PKA‐C (Thr197) (1:1000; Cell Signaling, #5661), GAPDH (1:1000; Cell Signaling, #5174) and Anti‐rabbit IgG, HRP‐linked Antibody (1:2000; Cell Signaling, #7074).