Odyssey clx digital imager

After transfections, cells were collected, lysed, and boiled in SDS sample buffer (1M Tris, 50% glycerol, 10% SDS, 0.5% bromophenol blue, β-mercaptoethanol). Samples were loaded onto SDS-PAGE gels and run. Proteins were wet transferred onto nitrocellulose membranes overnight (BioRad). Membranes were dried overnight and rehydrated in 1X TBS for 5 minutes. The membranes were subsequently blocked in Odyssey Blocking Buffer (LI-COR) for 1 hour at RT. Following blocking, membranes were incubated for 1–2 hours at RT with primary antibodies diluted in Odyssey Blocking Buffer + 0.2% Tween (VWR). Next, membranes were washed 4 × with TBS-T (0.1% Tween 20) for 5 minutes. Membranes were then incubated with secondary antibodies diluted in Odyssey Blocking Buffer + 0.2% Tween 20 at RT and protected from light. Following incubation with secondary antibodies, membranes were then washed 4 × with TBS-T for 5 minutes and rinsed with TBS. Membranes were imaged on an Odyssey CLx Digital Imager (LI-COR). Quantification of band signal intensities were performed using Image Studio Lite software (LI-COR).

For Western blot, lysates were collected on-plate with 1× radioimmunoprecipitation assay buffer (EMD Millipore, #20-188) with protease and phosphatase inhibitors (Thermo Fisher Scientific, #1861280). Lysates were sonicated and cleared before quantification with a Bradford Protein assay (Bio-Rad) and loaded at 50 μg per lane and run on a NuPage bis-tris gel (Thermo Fisher Scientific) and transferred to nitrocellulose membrane with the iBlot semidry transfer system (Thermo Fisher Scientific) and blocked in 5% milk in Tris Buffered Saline + Tween 20 (TBST) before staining with fibronectin (BD Biosciences, #610078; 1:10,000), ZEB1 (LSBio, #LS-C288694; 1:2000), E-cadherin (BD Biosciences, #610182; 1:1000), vimentin [Cell Signaling Technology (CST), #5741; 1:2000], RUNX1 (CST, #4336; 1:2000), RUNX2 (CST, #12556; 1:2000), RUNX3 (CST, #9647; 1:2000), CBFb (Abcam, ab33516; 1:2000), Snail (CST, #3879; 1:1000), Twist1/2 (Abcam, ab50887: 1:50), and CoxIV (CST, #11967; 1:2000) overnight in 5% milk in TBST. LI-COR secondary antibodies, IRDye goat anti-rabbit and goat anti-mouse, 800CW (LI-COR, #925-32219), and 680RD (LI-COR, #925-68076), were applied at 1:10,000 for 1 hour at room temperature in 5% milk in TBST before imaging on the LI-COR Odyssey CLx Digital Imager.