2.4. Western Blot

Western blot analysis was performed as described previously [27]. In brief, the cells were seeded at 5 × 10⁵ cells per well (6 well plates, Greiner-Bio-one) and incubated with the individual drugs as described above at 37 °C and 5% CO₂. Cells were washed twice with Dulbecco’s Phosphate Buffered Solution (DPBS, Gibco) and harvested in ice-cold lysis buffer (Ripa buffer, Thermo Scientific, supplemented with Protease Inhibitor Cocktail, Thermo Scientific). The lysates were homogenized, and the protein content was determined enzymatically (BCA Protein Assay, Thermo Scientific). An SDS sample buffer (4× Laemmli Sample Buffer, BioRad, supplemented with Bond Breaker, Thermo scientific) was added, and heat denaturation was performed at 95 °C for 3 min, except for the probes for CXCR4, which must not be boiled. Twenty micrograms of the samples were loaded on SDS-polyacrylamide gels (10% polyacrylamide) and size fractionated by electrophoresis.

For immunoblotting, the proteins were transferred onto immobilon-P PVDF transfer membranes (Merck, Millipore, Burington, MA, USA). The membrane was blocked with 5% nonfat dried milk (BioRad) in Tris Buffer Solution (TBS) (140 mM NaCl, 10 mM Tris-HCl, pH 7.4) and incubated with the primary antibodies (for details, see Supplementary Table S1) for 2 h at room temperature or overnight at 4 °C. After intense washes with TBS, secondary antibodies (for details, see Supplementary Table S1) were added and incubated for 1 h. Bound antibodies were detected with a chemiluminescence detection system (ECL Clarity Western ECL Substrate, BioRad).