Figure 7:

4F-induced elevation of apoE secretion requires ABCA1. Immortalized apoE3 TR astrocytes were transfected with an ABCA1-specific CRISPR/Cas9 vector and a homology directed repair (HDR) vector for puromycin resistance and red fluorescence protein (RFP) to establish a stably transfected ABCA1 KO cell line. (A) Transfection is confirmed by continuous expression of RFP. Un-transfected apoE3 TR cells (WT, normal ABCA1 expression) show no RFP signal. (B) ABCA1 WT and ABCA1 KO apoE3 TR astrocytes were treated for 6 hours with or without 5μM 4F in serum-free OPTI-MEM. SDS-PAGE was performed on samples to determine the relative amount of apoE secreted, as well as ABCA1 protein expression levels. Tubulin was used as a loading control. Results were obtained from n=3 independent experiments with each treatment in duplicate. * = p < 0.05; NS, not significant.