Anti icam 1

Pulmonary endothelial cells or lung tissue were lysed with RIPA buffer. Proteins were separated by electrophoresis in a denaturing polyacrylamide gel and transferred to a PVDF membrane. After blocking with 5 % milk-Tris-buffered saline and Tween 20 (TBST) and washing in TBST, membranes were then incubated in the appropriate primary antibodies (anti-ICAM-1; both from R&D, Emeryville, USA) and anti-GAPDH (Cell Signal Technology, Beverly, USA) at 4 °C overnight. After washing, membranes were incubated with the appropriate HRP-conjugated secondary antibodies and analyzed by ECL development.

Plumericin was isolated from bark material of Himatanthus sucuuba, as described earlier66 (link). PHA-408 was obtained from Axon Medchem BV (Groningen, Netherlands) and NAC, DPI, Triton X-100, medium M199, fetal bovine serum (FBS), goat serum, gelatin, and paraformaldehyde (PFA) were purchased from Sigma Aldrich (Saint Louis, USA). Penicillin, streptomycin, fungizone, and trypsin-EDTA were bought from LONZA (Visp, Switzerland), endothelial cell growth supplement (ECGS) with heparin from PromoCell (Heidelberg, Germany), and human recombinant TNFα from PeproTech (Vienna, Austria). The products 2′,7′-dichlorodihydrofluresceindiacetate (H2-DCF) and TMB ELISA substrate were obtained from ThermoFischer Scientific (Vienna, Austria). Hoechst 33342 and Alexa 555 were obtained from Life Technologies (Columbus, USA), mouse IgG HRP-linked antibody from GE Health care (Little Chalfont, UK), anti-CD31 and anti-p16 from BioLegend (San Diego, USA), anti-p65 from Santa Cruz (Santa Cruz, USA), anti-Ki-67 from ThermoFischer Scientific, anti-p21 from BD Bioscience (Franklin Lakes, USA), anti-E selectin and anti-ICAM-1 from R&D Systems (Abingdon, UK). Trizol, MuLV-reverse transcriptase, RNAse inhibitor and, oligo dT primers were obtained from Life Technologies, and FastStart SYBR Green Master Mix from ThermoFischer Scientific.

Samples (cells or tissues) were washed twice with PBS and fixed in ice-cold acetone for 5 min at room temperature, air dried, rehydrated in PBS and then blocked for 1 h in Dako Serum-Free Protein Block (DSFPB) (Agilent Technologies, Santa Clara, CA). Samples were incubated with purified primary antibodies (or suitable isotype matched controls) in DSFPB for 2 h at room temperature, washed in PBS, and then probed with Alexa-Fluor 647 labeled anti-Murine IgG raised in goat (Thermo Fisher Scientific) for 30 min. Following washing in PBS, samples were stained with fluorophore-conjugated primary antibodies for 2 h at room temperature if required. Samples were washed in PBS, counterstained with DAPI for 10 min and mounted with a coverslip using Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Immunofluorescence images were acquired using a Zeiss Cell Observer SD confocal fluorescence microscope (Zeiss, Oberkochen, Germany). Fluorochrome-labeled antibodies: AF594 anti-CD31 (catalogue number 303126; BioLegend, San Diego, CA), AF647 anti-mouse IgG raised in goat (catalogue number A21235; Thermo Fisher Scientific). Unconjugated antibodies for IFM: anti-ICAM-1, anti-VCAM-1, anti-P-selectin and IgG1 isotype control (catalogue numbers BBA3, BBA5, BBA30, MAB002 respectively; R&D, Abingdon, UK).