Elk 1

The array slides (breast cancer BR20834 and TNBC BR1503b) were purchased from US Biomax Inc. (Rockville, MD, USA). The BR20834 slides included 205 invasive ductal carcinomas, 2 invasive lobular carcinomas, and 1 invasive papillary carcinoma. However, 15 invasive ductal carcinomas were lost during the evaluation. The BR1503b slides included 7 breast intraductal carcinoma and 60 breast invasive ductal carcinoma, which contained 30 TNBC, duplicate cores per case. Detailed information for this array can be viewed at http://www.biomax.us/tissue-arrays/. The sections were immunostained for PKCα (1:200) (BD Biosciences, San Jose, CA, USA), Elk-1 (1:400) (Santa Cruz, CA), and MZF-1 (1:400) (Santa Cruz) and the expression was scored by staining as follows: 1+, weak; 2+, moderate; and 3+, strong.

Total proteins from PMVECs and lung tissues were isolated using radio-immunoprecipitation assay lysis (20–188, Merck, Zurich, Switzerland), quantified using BCA, boiled in sample preparing buffer to prepare samples, and separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The separated proteins were transferred to PVDF membranes, which were sealed in 3% skimmed milk for 30 min and probed with diluted primary antibodies overnight at 4 °C. On the next day, PVDF membranes received washing and 1-h probing with HRP-coupled secondary antibody (1:5000, ab205719, Abcam) at room temperature. The used antibodies are as follows: Fcgr2b (1:1000, 550,270, BD Biosciences), vascular endothelial (VE)-cadherin (1:3000, sc-9989, Santa Cruz Biotechnology), β-catenin (1:3000, 13-8400, Invitrogen), Elk1 (1:1000, sc-365,876, Santa Cruz Biotechnology), GAPDH (1:5000, ab8245, Abcam).

At specified time intervals and after serum starvation and/or transient transfection, immortalized mouse carotid VSMC were harvested with PBS and pelleted by centrifugation for 10 min in a JS-4.2 rotor at 3000 rpm. The pellet was resuspended in hypotonic buffer (20 mM HEPES, pH 7.9, 25% glycerol, 1.5 mM MgCl2, 0.8 M KCl, 0.2 mM EDTA, 0.2 mM PMSF, 0.5 mM DTT) and incubated on ice for 30 min. Cell lysis was confirmed with microscopy and the cytoplasmic extract was removed by centrifugation at 18,000 RCF for 30 min at 4’C. The cell extracts were normalized using Lowry method and loaded on a 10% Tris Glycine gel and subjected to electrophoresis for 90 min at 125 V. Proteins were transferred to PVDF membranes and blocked in 5% milk with 1% BSA. The membrane was incubated with primary antibody (Elk-1 (1:1,000, Santacruz, sc-355), c-Myb (1:500, Santacruz, sc-517), sm22alph (1:1000, Santacruz, sc-53015) or GAPDH (1:10,000, Santacruz, sc-47724)) overnight at 4’C. The membranes were washed and incubated with donkey anti rabbit IgG (BioRad, #1706515) for 1 h at RT. The membranes were again washed and then incubated with Western ECL Lighting (Perkin-Elmer) for 1 min and detected on MiniBis Pro (Frogga Bio).