Anti sepp1

To determine protein expression, cells were harvested in RIPA buffer containing a protease inhibitor cocktail, and total protein was quantified using a bicinchoninic acid kit (Pierce, Rockford, IL, USA). Aliquots containing 8 μg total protein were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and then electro-blotted onto a 0.45-μm PV membrane (Immobilon™; Merck Millipore, Darmstadt, Germany). The membranes were blocked and probed overnight with the primary antibodies anti-SEPP1 (1:1000, #ab193193; Abcam, USA), anti-MyoD (1: 1500; Invitrogen, Carlsbad, CA, USA), anti-MyoG (1: 1500; Invitrogen, Carlsbad, CA, USA), anti–β-catenin (1:5000, #ab32572; Abcam, USA), and anti–active β-catenin (1:500, #05-665; Merck Millipore).

Colonoids of TgM9 and their WT littermates’ mice grown on Matrigel in a 24 well plate were washed with room temperature 1X PBS. Then cold 1X PBS was added to disrupt the colonoids. Disrupted colonoids were centrifuged at 5000 rpm for 5 min at 4 °C. After carefully removing and discarding the supernatant (mix of PBS and Matrigel), colonoid-pellet was lysed with cell lysis buffer RIPA^{11 (link)}] and protein estimation was done. Colonoid lysates (10 µg for day 4 and 20 µg for day 7 and day 9) were used for the gel electrophoresis. The primary antibodies used were anti-CA1 (kind gift from Dr A. Waheed; Department of Medicine, St. Louis University, St. Louis, MO, USA), anti-PCNA (Abcam), anti-SEPP1 (Abcam). Goat anti-rabbit secondary antibody (Abcam) was used. Equal loading of the WBs was normalized by using anti-GAPDH as the loading control.