Epsins Regulate Mouse Embryonic Stem Cell Exit from Pluripotency and Neural Commitment by Controlling Notch Activation

Figure 2

EPN1 and EPN2 silencing in mESCs impairs Notch pathway activation. (a) EPN1 and EPN2 KD does not affect clathrin levels. Western blot assay shows comparable clathrin levels in shCTRL and shEPN cells. β-Actin was used as the loading control. (b) EPN KD does not affect clathrin-mediated endocytosis. Representative images of clathrin-mediated uptake of Alexa488-transferrin in shCTRL and EPN1 and EPN2 KD cultures. Nuclei are counterstained with Hoechst 33258. (c) EPN KD impairs Notch activation. EPN KD abolishes uptake of N1FC and reduces NICD levels. Nuclei are counterstained with Hoechst 33258. β-Actin was used as the loading control. (d) EPN1 and EPN2 KD result in reduced expression levels of Notch-regulated target genes. qRT-PCR analysis of HES1 and p21 in shEPN1 and shEPN2 KD mESCs. Levels are normalized over the control line (shCTRL), using β-actin as the housekeeping gene. All data are expressed as the means ± STDV ( biologically independent experiments). Statistical significance (unpaired -test): and .

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