Agilent 6100 lc ms spectrometer

Phenylephrine was purchased from Sigma-Aldrich (St. Louis, MO, USA) and CXCL12 from Protein Foundry, Milwaukee, WI, USA. siRNA reagents were purchased from GE Dharmacon (Lafayette, CO, USA). The peptide analogs of transmembrane helix 2 (TM2) (LLFVITLPFWAVDAVANWYFGNDD-NH₂) and TM4 (VYVGVWIPALLLTIPDFIFANDD-OH) of CXCR4 were generated using the Fmoc protected amino acids in a solid-phase synthesis on a 433A Applied Biosciences Peptide Synthesizer and Liberty Blue Microwave peptide synthesizer (CEM Corporation, Charlotte, NC, USA) using Fmoc chemistry. The peptides were cleaved from the resin and deprotected with a mixture of 90.0% (v/v) trifluoroacetic acid (TFA) with 2.5% water, 2.5% triisopropyl-silane, 2.5% 2,2′-(ethylenedioxy)diethanethiol and 5% thioanisol. Peptides were purified on a preparative (25 mm × 250 mm) Atlantis C3 reverse phase column (Agilent Technologies, Santa Clara, CA, USA) in a 90 min gradient of 0.1% (v/v) trifluoroacetic acid in water and 0.1% trifluoroacetic acid in acetonitrile, with a 10 mL/min flow rate. The fractions containing peptides were analyzed on Agilent 6100 LC/MS spectrometer (Agilent Technologies, Santa Clara, CA, USA) with the use of a Zorbax 300SB-C3 PoroShell column and a gradient of 5% acetic acid in water and acetonitrile. Fractions that were more than 95% pure were combined and freeze dried.