Pka cα

HEK-293 cells expressing recombinant FLAG-CPEB3a protein were harvested 36 h post-transfection by scraping in ice-cold lysis buffer containing 10 mM HEPES pH 7.4, 200 mM NaCl, 2 mM EDTA, 1% Tx-100, Complete Mini protease inhibitors (1 tablet/10 ml) and Phos-Stop phosphatase inhibitors (1 tablet/10 ml). Lysates were sonicated for 10 min in ice-cold sonication bath and spun down (15 min/17.000 rcf). Supernatants were mixed with 50 μl of FLAG M2 agarose beads (Sigma), previously blocked with 3% BSA, and incubated for 2 h at 4°C on a rotator, followed by 5x washing with lysis buffer. CaMKII was preactivated at 30°C for 30 min in kinase reaction buffer (New England Biolabs) supplemented with 1.2 μM calmodulin and 2 mM CaCl₂. Beads were mixed with kinase buffer containing 1 mM ATP and PKA-C_α (5000 U, New England Biolabs), pre-activated CaMKII (1000 U, New England Biolabs), or the corresponding buffer without enzyme. Sample was incubated for 30 min at 30°C with rotation and then mixed with 50 μl of NuPage reducing sample buffer and separated by SDS PAGE.

Drugs were prepared as frozen stock solutions (stored below −20 °C). Compounds were as follows: N,N,H,-Trimethyl-5-[(tricyclo[3.3.1.13,7]dec-1-ylmethyl)amino]-1-pentanaminium bromide hydrobromide (IEM-1460; Hello Bio); 4-(3-(cyclopentyloxy)-4-methoxyphenyl)pyrrolidin-2-one (rolipram; Abcam); 4-[(2 S)-2-[(5-isoquinolinylsulfonyl)methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl] phenyl isoquinolinesulfonic acid ester (KN-62; Tocris and Hello Bio); D-AP5 (Hello Bio); L-689,560 (Tocris); a catalytic subunit of protein kinase A (PKA Cα, New England Biolabs); Ca²⁺/calmodulin-dependent protein kinase II (CaMKII, New England Biolabs).