Secondary anti mouse antibody

All cell lines were cultured on sterile glass slides. The cells were then fixed with Roti-Histofix 4% (Carl Roth, Karlsruhe, Germany) for 15 min, washed with PBS (Sigma/Merck, Darmstadt, Germany), blocked with 5% milk (Th. Geyer, Berlin, Germany) in PBS and permeabilized with 0.01% Triton X-100 (Carl Roth). Incubation with the anti-MUC1 mouse antibody (Cell Signaling) was performed overnight at 4 °C. Direct FITC-labeled anti-EpCAM (Acris) and anti-pan cytokeratin (CK8, CK18, CK19, Abcam) antibodies, as well as the secondary anti-mouse antibody (Dianova), were applied the next day for 1 h at RT. Cell nuclei were visualized using Hoechst 33258 (Sigma, Darmstadt, Germany). Images were taken using an inverted fluorescence microscope at 20× magnification (Carl Zeiss Microscopy).

Cultured U-937wt cells were treated with BT44 at a concentration of 50 μM, then 2 μM of etoposide was administered 6 h later. After 15 h of incubation, cells were lysed in RIPA buffer, separated by PAGE on a 15% polyacrylamide gel and transferred onto a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with PBS containing 5% (w/v) skimmed milk and incubated with a primary antibody against Caspase-3 (Cell Signaling, Danvers, MA, USA) and secondary antibodies (Abcam, Cambridge, UK) at room temperature for 1 h. Band intensity was quantified using the ChemiDocTM system (Bio-Rad, Hercules, CA, USA). To assess the content of Hsp70 in treated cells, 20 μg of the same cell lysates were separated by PAGE on an 11.5% gel. The membrane was probed with an anti-Hsp70 monoclonal antibody (Clone 2H9) followed by a secondary anti-mouse antibody (Abcam).

Antibodies for detection of AAT and secondary anti-mouse antibodies were obtained from Abcam (Cambridge, UK). HNE, together with a bicinchoninic acid (BCA) protein assay kit were obtained from Thermo Scientific (Rockford, IL, USA). The standard p-nitroaniline (p-NA) and the peptide substrates used for the determination of human HNE and Cat. G activities (MeOSuc-Ala-Ala-Pro-Phe-NA and Suc-Ala-Ala-Pro-Phe-NA, respectively) were from Bachem (Bachem AG, Bubendorf, Switzerland). Unless otherwise stated, all other analytical grade reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Double-distilled water used for the preparation of all buffers was prepared with a Millipore (Bedford, MA, USA) Milli-Q purification system.