The plate was incubated for 60 min at room temperature, washed four times, incubated for 30 min with HRP-conjugated anti-rabbit IgG, again washed, and incubated with tetramethylbenzidine (TMB) substrate. After 1 h, the stop solution was added and A450 nm measured. A standard curve was generated using purified PKA provided by the manufacturer. pCREB, CREB, and β-tubulin immunoblotting for PKA activity Postconfluent HMVEC-Ls were exposed to ET (1000 ng/mL:1000 ng/mL), ET + H-89 (10 μM), ET + KT-5720 (10 μM), FSK (10 μM),
IBMX (1 mM), or medium alone, after which they were lysed with ice-cold modified radioimmunoprecipitation assay buffer, containing 50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 100 mg/ml type-1 DNase, 1 mM sodium orthovanadate, 1 mM CP-868596 cell line NaF, 1 mg/ml pepstatin A, 10 mM pyrophosphate, and 1 mM phenylarsine oxide (all purchased from Sigma), and 1 tablet of NSC 683864 complete Fludarabine cost protease inhibitor mixture (Roche Applied Science) per 20 ml of lysate as described . The lysates
were centrifuged, and the supernatants were assayed for protein concentration with a Bradford protein assay kit (Bio-Rad). The samples were resolved by 8-16% gradient SDS-PAGE and transferred onto PVDF membranes. The blots were blocked with membrane blocking solution (Zymed Laboratories Inc., San Francisco, CA) and were incubated with biotinylated rabbit anti-pCREB antibodies (Cell Signaling), followed by streptavidin HRP (Cell Signaling), after which they were developed with enhanced chemoluminescence (ECL). To control for protein loading and transfer, the blots were stripped and reprobed with either murine anti-CREB and/or murine anti-β-tubulin (Invitrogen), and each pCREB band was normalized to total CREB and/or β-tubulin signal in the same lane on the same blot. Statistics One-way analysis of variance, followed by post hoc comparisons using Tukey-Kramer’s multiple pairwise comparison test, was used to compare the mean responses
among experimental and control groups for all experiments. SAS 9.2 was used for the analyses (SAS Institute Inc., Cary, NC, USA). BCKDHA A p value of < 0.05 was considered significant. Acknowledgements This work was supported in part by grant HL089179 from the NIH (SEG) and MARCE (ASC). We would also like to thank Lei Zhang, MD, and Grish Ramachandra, PhD, for assisting in the purification of PMNs. Electronic supplementary material Additional file 1: Figure S1. FSK and IBMX do not reproduce the ET effect on IL-8-driven TEM of PMNs at 0.5 h. (A) HMVEC-Ls were treated for 0.5 h with FSK (10 μM), IBMX (1 mM), or medium alone, and lysed. The lysates were processed for pCREB immunoblotting. IB, immunoblot, IB*, immunoblot after stripping. To control for protein loading and transfer, blots were stripped and reprobed for β-tubulin.