Sc 822

Western immunobloting analysis of IL-6, IL-8, CXCR2, SOCS-3, p-STAT-3, p-JAK2 and p-c-Jun expression was also performed on five RCC samples. After homogenization and fractionation of fresh frozen tumor tissue, 100 μg protein was separated on a 10% polyacrylamide gel and blotted onto nitrocellulose membranes, probed with primary antibody overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated goat-anti-rabbit IgG or HRP-conjugated goat-anti-mouse IgG secondary antibody (AP132P and AP124P respectively, Chemicon, Millipore, Temecula, CA, USA). The same primary antibodies described in Table 2 were used at the following dilutions: 1:2,000 for anti–p-STAT-3, 1:200 for anti–SOCS-3, anti-CXCR2, and anti-IL-6. The anti-IL-8 antibody was diluted to a concentration of 0.1 μg/mL. The anti-p-JAK2 (sc-16566-R, Santa Cruz, 200 μg/ml) and anti-p-c-Jun (sc-822, Santa Cruz, 200 μg/ml) were diluted at 1:200. Bands were visualized using ECL chemiluminescence detection reagents (Perkin Elmer, Athens, Greece). Relative protein amounts were evaluated by a densitometric analysis using Image J software (La Jolla, CA, USA) and normalized to the corresponding Actin levels. All experiments have been performed at least 3 times and representative results of one experiment are shown.

Protein was extracted from HBE cells according to previous procedures 18 . The following antibodies were used to determine the expression of corresponding protein: ITGB4 (ab197772, Abcam, USA), EGFR (ab52894, Abcam, USA), p-EGFR (ab32430, Abcam, USA), ERK1/2(ab184699, Abcam, USA), p-ERK1/2(ab223500, Abcam, USA), c-Jun (sc-74543, Santa Cruz, USA), p-c-Jun (sc-822, Santa Cruz, USA). Lamin-β1(ab133741, Abcam, USA) and β-actin (ab8226, Abcam, USA) were used as corresponding controls, respectively.

Protein extraction from HBECs was performed according to previous procedures.²⁶ In brief, 50 µg protein was isolated and separated from HBECs by 10% SDS‐PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. Then, the PVDF membrane was incubated with primary antibody for 12 hours and next incubated with Horseradish Peroxidase (HRP) conjugated secondary antibody. Expressions of c‐Jun (Santa Cruz, sc‐74543) and phosphorylated c‐Jun (Santa Cruz, sc‐822) were determined with corresponding antibodies.