Phospho jnk

Fluorescein isothiocyanate (FITC)-conjugated CD90 and CD45 antibodies and puri.ed anti-rat
antibody to CD73 were purchased from BD Biosciences Pharmingen (San Diego, CA, USA).
FITC-conjugated CD11b antibody was bought from GeneTex, Inc. (USA). FITC-conjugated CD31 and
44 antibodies were purchased from Serotec (Oxford, UK). Bax and Bcl-2 antibodies were bought
from Abcam (Cambridge, UK). Horseradish peroxidase (HRP)-conjugated secondary antibody,
caspase-3, β-actin, c-Jun N-terminal kinase (JNK), P38, Phospho- JNK and P38 antibodies were
obtained from Cell Signaling (Danvers, MA, USA). 2, 4, 6-tri (2-pyridyl)-striazine (TPTZ)
was bought from Merck (Germany) and 3-(4 , 5 (link)-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium
bromide (MTT), streptozotocin (STZ), collagenase type I and 2, 7-dichlorofluorescein
diacetate (DCF-DA) were bought from Sigma (Sigma Aldrich, St. Louis, MO, USA). All cell
culture materials were obtained from Gibco (Carlsbad, CA, USA).

The standard reference airborne PM (standard reference material 1649b), mainly composed of polycyclic aromatic hydrocarbons, polychlorinated biphenyl congeners, pesticides, and doxins, was purchased from the National Institute of Standards and Technology (Gaithersburg, MD, USA). The specific molecular inhibitors SP600125, U0126, LY294002, AH6809, and NS398 were purchased from Selleck (Houston, TX, USA). Antibodies against COX-2, phospho-ERK, ERK, phospho-JNK, JNK, phospho-AKT, AKT, and EpCAM were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-Filaggrin antibody was obtained from GeneTex (San Antonio, TX, USA) and anti-GAPDH antibody was purchased from Beyotime (Shanghai, China). mRNA primers were synthesized by Sangon Biotech (Shanghai, China). The reagents for real-time qPCR were purchased from TaKaRa Bio (Shiga, Japan). The reagents used for western blotting were obtained from Solarbio Life Science (Beijing, China).

To identify the important roles of the NRP1 and MAPK signaling pathways in the proangiogenic function of Tagln2, the expression levels of NRP1, two major MAPK signaling pathway molecules (extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK)), and some differentially expressed proteins or markers involved in angiogenesis, migration and apoptosis were measured. The expression levels of NRP1 and TAGLN2 were modulated with siRNA or plasmid vector in Ealy926 cells stably expressing TAGLN2 and control cells, as appropriate. Then, the corresponding functional assays, including cell migration and tube formation assays, were conducted. EC samples (Ealy926 cell) with TAGLN2 or NRP1 expression modulation were harvested and prepared for western blot analysis. Blots were incubated with primary antibodies against Tagln2, VEGFR2, VWF, E-Cadherin, NrCAM, P-selectin, Caspase 3/cleaved Caspase 3, PARP1/cleaved PARP1 (ProteinTech, Cat#15508-1-AP, 67407-1-AP, 11778-1-AP, 60335-1-AP, 21608-1-AP, 60322-1-AP, 19677-1-AP and 13371-1-AP), NRP1, Phospho-ERK1/2, Total ERK1/2 (Abcam, Cat#ab25998, ab50011 and ab54230), Phospho-JNK, and Phospho-C-JUN (Cell Signaling Technology, Cat#4668 and 3270), respectively. Targeted proteins were detected with enhanced chemiluminescence (ECL, Millipore, USA).

Dulbecco's modified Eagle's medium (DMEM) was obtained from Life Technologies (Grand Island, NY, USA); Fetal bovine serum (FBS) from PAN Biotech (GmbH, Aidenbach, Germany); Hank's Balanced Salt Solution (HBSS), Epidermal Growth Factor (EGF) and Insulin, Transferrin, Selenium, Sodium Pyruvate solution (ITS-A) from Invitrogen; Antibodies against phospho-p38, phospho-ERK1/2, phospho-JNK, beta-actin and caspases were purchased from Cell Signaling (Beverly, MA, USA) and antibodies against poly ADP ribose polymerase (PARP) was from Santa Cruz Biotechnology (Santa Cruz, CA). [6]-gingerol was purchased from Biomol (Hamburg, Germany). The MAP kinase inhibitors U0126, SP600125, SB203580 and NF-kappaB inhibitor SN50 were procured from Calbiochem (San Diego, CA). All other chemicals, including Phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma Chemicals (St. Louis, MO, USA).