Anti gp130

HBMEC cultures were washed with PBS, and protein was harvested by lysing the cells with Radio Immuno Precipitation Assay (RIPA) buffer containing Complete Mini Protease Inhibitor Cocktail (Roche). HMC3 were washed with PBS, with additional steps performed to isolate the nuclear and cytoplasmic fractions as described earlier [28 (link)].
Protein concentrations were quantified using a BCA Protein Assay Kit (ThermoFisher Scientific, ref #23223) and immunoblotting was performed as described previously [6 (link)]. The following primary antibodies were used: anti-gp130 (IL6ST; Abcam, ab283685), anti-IL-6R (Abcam, ab128008), anti-phosphorylated STAT3 (Tyr705, Cell Signaling, 9145T), and anti-STAT3 (Cell Signaling, 124H6). In addition, the following secondary antibodies (all from Licor) were applied: IRDYE^® 680RD (926-68073 + 926-68072) and IRDYE^® 800CW (926-32212 + 926-32211).
Protein levels were normalized to anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Novus, NB3000-221R) for total and cytoplasmic fractions, and to anti-Lamin A/C (Cell Signaling, #4777S) for nuclear fractions. Membranes that were used to read multiple proteins were stripped using NewBlot Nitro Stripping Buffer (Licor, ref #928-40030) for 10 min, then the steps were repeated starting with the blocking of the membrane.

Cells were harvested and lysed in PRO-PREP protein extraction solution (iNtRON, Bio Inc, Sungnam, Korea). Protein concentrations were measured with a Bradford Protein Assay Reagent kit (Bio-Rad, Richmond, CA, USA). Proteins were fractionated by 10% SDS-polyacrylamide gels electrophoresis (PAGE), and transferred onto polyvinylidene difluoride (PVDF) membranes. These were incubated with anti-PCNA, anti-cyclin D1, anti-Bcl-2, anti-phospho-JAK2, anti-β-actin Ab (1:1000; Abcam), anti-gp130, anti-phospho-STAT3 and anti-iNOS antibodies (1:1000; Cell Signaling Technology, Beverly, MA, USA) as primary antibodies. Goat anti-rabbit horseradish peroxidase-conjugated IgG or goat anti-mouse horseradish peroxidase-conjugated IgG (Abcam, Cambridge, MA, USA) served as secondary antibodies. Protein bands were detected with a chemiluminescence reagent kit (SurModics, MN, USA). The data are expressed as means ± SD of three independent experiments.