A21058

Western analysis of MCC progenitors was performed by generating mutant and RNA-injected embryos as described above and then isolating the skin progenitors at stage 10.5 by removing the animal cap, trimming away marginal zone tissue, and culturing in 0.5× MMR. Multicilin-HGR was activated by treating with dexamethasone at stage 11, which is sufficient to drive most skin progenitors (~95%) into MCC differentiation (18 (link)). At the indicated stage equivalent, the animal cap tissue was collected and directly lysed with rigorous pipetting and vortexing in 2× lithium docecyl sulfate (LDS) sample buffer (Thermo Fisher Scientific, NP0008), followed by boiling for 10 min. The sample proteins were subjected to SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes that were then blocked with 0.1% casein blocker (#161-0783, Bio-Rad) in 0.5× PBS for 30 min followed by overnight incubation with the indicated primary antibodies at 4°C in 0.1% Tween-20 in blocking buffer. After extensive washing in 0.1% Tween-20 in PBS, the blots were incubated with Alexa Fluor 680– or 800–conjugated secondary antibodies (Thermo Fisher Scientific, A21058, A21076, and A32735) for 45 min at room temperature, washed with 0.1% Tween-20 in PBS, and imaged using Odyssey (LI-COR) instrument. The primary antibodies used in this study are listed in table S6.