Csf 1

MDA-MB-231 cells were seeded on top of either compliant (1kPa) or stiff (10kPa) gels for 24 hours and treated with or without the FAK inhibitor PF573228 (MilliporeSigma). Cells were rinsed with 1X PBS and lysed with 4X SDS sample buffer (4X Tris-Cl/SDS, pH6.8, 30% v/v glycerol, 10% w/v SDS, 0.09% v/v 2-mercaptoethanol, and 0.012% w/v Bromophenol Blue). Standard SDS-PAGE was conducted usingBio-Rad Any kD Mini-PROTEAN (4569035; Bio-Rad gels and PVDF membranes (Bio-rad). Membrane washing steps were performed with 0.1% polyoxyethylene 20 sorbitan monolaurate (Tween; JT Baker, Phillipsburg, NJ) in Tris-buffered saline. Blocking was performed with 5% milk in the washing buffer. Primary antibodies (GAPDH Biolegend poly6314; CSF-1 Santa Cruz sc-365779) were diluted in blocking buffer at 1:1000 dilution and applied to the membranes overnight at 4°C. Horseradish-peroxidase conjugated secondary antibodies were applied to the membranes in blocking buffer at 1:2000 dilution for 1 hour at room temperature. Membranes were imaged using SuperSignal chemiluminescent substrate and a FujiFilm ImageQuant LAS-4000. Quantification of protein expression was normalized to GAPDH loading control and densitometry was performed using Fiji.