Myristoylated pki 14 22 amide

Cells were maintained in modified Dulbecco’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 μg/mL of streptomycin, and 100 IU/mL of penicillin in 5% CO₂ at 37 °C and grown to ∼80% confluency before transfection with plasmids as described above. Small-molecule inhibitor rpcAMPs (cAMPs-Rp triethylammonium salt, Tocris, Cat. #1337) (370 μM) was added with transfection (36 h of total treatment) or after 12 h of transfection (24 h of total treatment); cell-permeable peptide inhibitor PKI (myristoylated PKI_14–22 amide, Tocris, Cat. #2546) (6.4 μM) was added 12 h after transfection (24 h of total treatment). The cells were harvested after a total of 36 h in transfection media (DMEM/10% FBS). Cells were briefly pulsed with 0.1 mM sodium pervanadate for 15 min at 4 °C prior to harvesting the cells to inhibit phosphatase activity and prevent peptide dephosphorylation to “lock” phosphorylation sites in place for downstream analysis. The cells were then washed twice with PBS, and pellets were frozen at −80 until further processing, as described in the Sample Preparation from Cell Lysates section.

The β₁-ADR partial agonist, xamoterol, the β₁-ADR antagonist, betaxolol and the protein kinase A (PKA) inhibitor, PKI 14-22 amide myristoylated (Tocris bioscience, Minneapolis, MN, USA) were used. Xamoterol was injected sub-cutaneously (3mg/kg). Betaxolol was injected sub-cutaneously (1mg/kg) or in the MeA (30nmole/side). PKI 14-22 amide was dissolved in 30% acetonitrile and injected in the MeA (2.75nmole/side) (Supplement 1).

Colon cancer cell lines Caco-2 (Catalog# HTB-37), HCT116 (Catalog# CCL-247), SW480 (Catalog# CCL-228) and SW620 (Catalog# CCL-227), and FHC (Catalog# CRL-1831) that serves as the control cell line were all purchased from the ATCC (American Type Culture Collection). FHC cells were cultured in RPMI-1640 medium supplemented with 20% FBS (Fetal Bovine Serum). HCT116, SW480, and SW620 cells were cultured in RPMI-1640 supplemented with 10% FBS, and Caco-2 cells were cultured in DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% FBS. Cell cultures were maintained at 37°C and supplemented with 5% CO₂. PDGF (Platelet-Derived Growth Factor) was purchased from Fisher Scientific (Waltham, MA). Forskolin (Catalog# F-9929) was from LC Laboratories (Woburn, MA). The biologically inactive Forskolin analogue (1,9-DideoxyForskolin from Coleus forskohlii) and the PKG inhibitor (Catalog# KT5823) were from Sigma-Aldrich (St. Louis, MO). Isoproterenol (Catalog# I6504), the general cAMP analogue 8-Br-cAMP, the specific PKA activator 6-Bnz-cAMP (Catalog# B4560), the specific Epac activator 8PCT-2Me-cAMP (Catalog# C8988), and the PKC activator PMA (Phorbol-12-Myristate-13-Acetate) were all from MilliporeSigma (Burlington, MA). The PKA inhibitor PKI 14–22 amide (myristoylated) and the Epac inhibitor ESI 09 were from Tocris Bioscience (Minneapolis, MN).