Analyzing Ptch1 Knockout MEFS Expression

Ptch1^−/− MEFS containing the integrated hPtch1 variants were trypsinized and analyzed compared to an uninfected (control) population using fluorescence activated cell sorting. A population of cells with fluorescence from the integrated hPtch1-mVenus-1D4 construct was clearly visible. This population was used to set a sorting gate for the cells containing the hPtch1 gene. Approximately 250,000 cells were sorted and then plated into a 6-cm dish. Once these cells were grown to a sufficient density, they were seeded into additional plates for Western blot analysis (41 (link)) to confirm expression of the hPTCH1-mVenus-1D4 gene. An anti–green fluorescent protein antibody (Novus Biologicals, catalog no. NB600-308, RRID: AB_10003058) was used to detect PTCH1 expression levels, and anti-P38 (Abcam, catalog no. ab7952, RRID: AB_306166) was used as a gel loading control.