Anti HA antibody was obtained from Covance , anti flag antibody was obtained from Sigma, and anti Xpress antibody was purchased from Invitrogen . Biotinylation of Anti Na ,K ATPase monoclonal antibody, 6H 6H was dialyzed against PBS at 4uC, diluted to 1 mg ml with PBS, and NaHCO3 was added to 50 mM, final concentration. EZ Link Sulfo NHS SS Biotin was mixed at 0.25 mg Biotin 1 mg 6H and samples were incubated overnight. Excess biotin was removed by dialysis against PBS. Biotinylated 6H had almost the same reactivity by Western blotting as non biotinylated 6H . Plasmid construction The A domain of the rat Na,K ATPase a subunit was amplified by polymerase chain reaction . This construct was subcloned as a BamHI EcoRI fragment into the pGEX 4T 3 vector to produce a cDNA encoding a GST fusion protein. The large cytoplasmic loop connecting the TM4 TM5 of the Na ,K ATPase a subunit was amplified by PCR with primers that included EcoR I and Not I restriction sites. The PCR fragment was subcloned into pGEX 4T 3 vector, in which the insert was fused to the carboxyl terminus of glutathione S transferase.
To generate deletions, BspEI, ClaI, MfeI and HindIII sites were introduced in the pGEX 4T3 construct by creating silent mutations. Mutated constructs were digested with NotI plus BspEI, NarI, ClaI, MfeI or HindIII for Cterminal deletions or EcoRI and ClaI for the N terminal deletion. Small fragments were removed by agarose gel electrophoresis, blunt ends were introduced with pfu DNA polymerase and the modified constructs Pazopanib selleckchem were recircularized by ligation. The H85N chimera, which is composed of the H ,K ATPase and the Na ,K ATPase . PP2A A subunit was also cloned by PCR with primers that included Kpn I and Xba I restriction sites, and sequence encoding a flag epitope tag. The PCR fragment was subcloned into the pcDNA 3.1 vector. The flag epitope was fused to the amino terminus of the PP2A A subunit construct. All PCR primer sequences are available on request. Arrestin 2 and 3, and GRK 2 and 3 were cloned by PCR from a human kidney cDNA library.
PCR was performed with Tivantinib primers that included Kpn I and Xba I restriction sites, and sequence encoding a flag or HA epitope tags. The flag and HA tags were fused to the amino terminus of the resulting constructs for arrestin 2 and 3, and to the carboxyl terminus for GRK 2 and 3. The PCR fragments were subcloned into the mammalian expression vector pcDNA 3.1 . Cell culture and transfection COS cells were cultured in a humidified incubator under 5% CO2 in a MEM supplemented with 10% FBS, 2 mM L glutamine, 50 U mL penicillin and 50 mg ml streptomycin. DNA transfection was performed with Lipofectamine 2000 according to the manufacturer?s instructions, and assays were performed 48 h after transfection.