Tgx fastcast acrylamide kit

Cells were washed 1x in PBS prior to harvesting in preheated, high-salt sample lysis buffer (20% glycerol, 2% sodium dodecyl sulfate (SDS), 30% 10X PBS, 2.5% β-mercaptoethanol). Scraped cells were transferred to Eppendorf tubes, heated at 95 °C for 1 min, pulled through an insulin syringe three times, and heated again. Samples were separated on 7.5% SDS-PAGE (Bio-Rad, Hercules, CA, USA; TGX FastCast Acrylamide Kit) using Tris-Glycine running buffer and transferred to a nitrocellulose membrane (GE) with a 0.45 μm pore size. Antibodies were diluted in Odyssey Blocking Buffer TBS (LI-COR) as follows: anti-APC-M2 Chicken pAb (1:2000), anti-β-catenin mouse mAb (1:1000), anti-phospho-Ser33/Ser37/Thr41-β-catenin rabbit pAb (Cell Signaling Technology, Danvers, MA, USA; 1:500), anti-α-tubulin DM1A mouse mAb (Santa Cruz Biotechnology, Dallas, TX, USA; 1:1000), anti-β-actin mouse mAb (Sigma, 1:1000), and IRDye 680LT and 800CW anti-rabbit, anti-mouse, or anti-chicken secondary antibodies (1:150,000). Immunoblots were imaged on an LI-COR Odyssey CLx imaging system.

Cells were washed 1x in PBS prior to harvesting in pre-heated high-salt sample lysis buffer (20% glycerol, 2% SDS, 30% 10x PBS, 2.5% β-mercaptoethanol). Scraped cells were transferred to Eppendorf tubes, heated at 95 °C for 1 min, pulled through an insulin syringe three times, and heated again. Samples were separated on 7.5% SDS-PAGE (Bio-Rad, TGX FastCast Acrylamide Kit) using Tris-Glycine running buffer and transferred to a nitrocellulose membrane (GE) with a 0.45-μm pore size. Antibodies were diluted in Odyssey Blocking Buffer TBS (LI-COR) as follows: anti-APC-M2 rabbit pAb (1:2,000), anti-β-catenin mouse mAb (1:1,000), anti-Axin1 goat (1:500, R&D Systems #AF3287), and anti-β-actin mouse mAb (Sigma) (1:1,000), IRDye 680LT and 800CW anti-rabbit or anti-mouse secondary antibodies (1:15,000). Immunoblots were imaged on a LI-COR Odyssey CLx imaging system.