The following antibodies were used: mouse monoclonal anti-Cpt1a (1:500(sorted), (1:1000crypt lysates), Abcam ab128568), rabbit monoclonal anti-HMGCS2 (1:500(sorted), 1:1000(crypt lysates), Abcam, ab137043), rabbit monoclonal anti-FABP1 (1:1000, CST, 13368), monoclonal rabbit anti-PDK4 (1:1000, Abcam, ab214938), rabbit monoclonal γ-tubulin (1:1000, Sigma-Aldrich, T5198) and monoclonal anti-rabbit total H3 (1:3000, CST 4449S).
Lgr5-eGFPhi ISCs, Lgr5-eGFPlow progenitors, or EpCAM+ cells were sorted directly into 6X Laemmli sample buffer (Alfa Aesar, J61337) and boiled for 5 minutes. Either 10,000 cells were loaded per sorted sample or 10ug of crypt lysates were loaded per sample onto a 4%–12% gradient gel, transferred on to PVDF membrane (Immobilon-P transfer, Millipore, ipvh00010), and analyzed using IgG-HRP antibodies (1:3000, CST, 7076, 7074) and Advansta WesternBright Sirus ECL detection kit (K12043D20).
Lgr5-eGFPhi ISCs, Lgr5-eGFPlow progenitors, or EpCAM+ cells were sorted directly into 6X Laemmli sample buffer (Alfa Aesar, J61337) and boiled for 5 minutes. Either 10,000 cells were loaded per sorted sample or 10ug of crypt lysates were loaded per sample onto a 4%–12% gradient gel, transferred on to PVDF membrane (Immobilon-P transfer, Millipore, ipvh00010), and analyzed using IgG-HRP antibodies (1:3000, CST, 7076, 7074) and Advansta WesternBright Sirus ECL detection kit (K12043D20).