Fig. 2. Cis-determinants required for LIN-12 downregulation. GFP (green), and AJM-1 (red) were visualized by immunofluorescence (see Materials and methods). Left panels show lateral views, with the apical surface toward the bottom. Right panels show ventral views, with the apical surface surrounded by AJM-1 staining. Throughout this study, the effect on downregulation was assessed in the daughters of the VPCs – the Pn.px stage – to ensure that P6.p has been induced and that there has been sufficient time for downregulation. (A,B) Schematic representation of VPCs at the Pn.px stage; green indicates LIN-12 accumulation in the apical domain, dashed lines the basolateral domain. Note that LIN-12 remains apical wherever it is detected in VPCs and their descendants. (C,D) LIN-12(+)::GFP is downregulated in the 1° lineage (P6.px), and can be seen at the apical membrane and in endocytic puncta (arrowheads) in the 2° lineage (P5.px and P7.px). (E,F) The DTS di-leucine is required for internalization and degradation. (G,H) The DTS serine/threonine residues are required for internalization and degradation. We note that these residues may regulate other aspects of LIN-12 trafficking or localization, as we sometimes see some basolateral accumulation of LIN-12(S/TtoA)::GFP (arrowheads in G). (I,J) LIN-12(KtoA)::GFP is internalized but not degraded in the 1° lineage; it accumulates in large and pleiomorphic endocytic puncta (arrowheads), suggestive of late endosomes (see also Fig. 4C). (K) Quantification of downregulation of different LIN-12 proteins. Scale bar: 10 µm.