Immunoblotting of HCT116 Cells from TME

Immunoblotting was performed on HCT116 beads from TME cultures as described in detail [41 (link)]. In short, HCT116 were retrieved from alginate beads by dissolving in 55 mM sodium citrate solution for 15 min and subsequent washing with Hank’s solution. Samples were lysed (50 mM Tris/HCl, pH 7.2/150 mM NaCl/(v/v) Triton X-100/1 mM sodium orthovanadate/50 mM sodium pyrophosphate/100 mM sodium fluoride/4 µg/mL pepstatin A/1 mM PMSF) for 30 min on ice to extract whole-cell proteins. Protein content was measured with the bicinchoninic acid system (Uptima, France) using bovine serum albumin (BSA) as standard, proteins reduced with 2-mercaptoethanol and total protein concentrations adjusted (500 ng per lane total protein). After separation of proteins with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), proteins were blotted onto a nitrocellulose membrane using a transblot apparatus (Bio-Rad, Munich). Membranes were incubated overnight (4 °C) with primary antibodies (1:10.000) and after subsequent washing, incubated for 2 h with alkaline-phosphatase coupled secondary antibodies (1:10.000). Finally, specific binding was detected using nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl-phosphate (VWR, Darmstadt, Germany) and bands quantified using the Quantity One program (Bio-Rad, Munich). β-Actin was used to normalize samples to control.