Vcam 1 sc 13160

Cells were lysed in 1% SDS lysis buffer. The BCA assay was used to determine protein concentrations. Proteins were separated using 10% SDS–PAGE gels. Proteins were then transferred onto polyvinylidene fluoride membranes. Nonfat milk in PBS was used to block the membrane at room temperature for 1 h. The membrane was incubated overnight at 4 °C with the primary antibody [p-P65 (ab76302; Abcam), P65 (8242; CST), ICAM-1 (sc-107; Santa Cruz), VCAM-1 (sc-13,160; Santa Cruz), and GAPDH (HC301; Transgen)]. After several washes with PBS, the membranes were incubated with the blocking buffer and secondary antibody coupled to horseradish peroxidase for 2 h at room temperature. The complexes formed on the membrane were then detected using ECLplus (Amersham Biosciences/GE Healthcare, Velizy, France).

Cell culture materials were purchased from Invitrogen (Carlsbad, CA, USA). Primary antibodies against p-STAT3 (Tyr705) (#9145, 1:1000), STAT3 (#30835, 1:2000), p-p65(Ser536) (#3033, 1:1000), p65 (#8242, 1:2000), and α-SMA (#19245, 1:2000) were purchased from Cell Signaling Technology (Beverly, MA, USA). Primary antibodies against IL-1β (sc-12742, 1:2000), CD34 (sc-7324, 1:2000), VCAM-1 (sc-13160, 1:2000), and EPAS-1 (sc-13596, 1:2000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against CD68 (28058-1-AP), ICAM-1 (60299-1-Ig, 1:2000), MCP-1 (66272-1-Ig, 1:2000), Bax (50599-2-Ig, 1:2000), Bcl-2 (12789-1-AP, 1:2000), cleaved-caspase-3 (19677-1-AP, 1:2000), GAPDH (10494-1-AP, 1:2000), FN1 (15613-1-AP, 1:2000), and SOX9 (67439-1-Ig, 1:2000) as well as secondary antibodies (Goat anti-mouse, SA00001-1; Goat anti-rabbit, SA00001-2) were purchased from Proteintech Group (Chicago, IL, USA). Recombinant human interleukin-1 receptor antagonist (IL-1Ra) (SRP3084) was obtained from Sigma (St. Louis, MO, USA), Stattic (inhibitor of STAT3) and Colivelin, (activator of STAT3) were purchased from MedChemExpress (Shanghai, China). Unless otherwise indicated, the remaining reagents used in this study were obtained from Sigma.