Figure 5

From: Caspase-mediated proteolysis of the sorting nexin 2 disrupts retromer assembly and potentiates Met/hepatocyte growth factor receptor signaling

Figure 5

Cleavage of SNX2 disrupts interaction with Vps35. (a) HeLa cells were transfected for 16 h with plasmids encoding Cherry-fused full-length SNX2 (WT), C-terminal fragment (Δ84) or N-terminal fragment (1–84). Cherry-fused protein integrity was analyzed by immunoblotting using an RFP antibody (top). Colocalization with early endosomes was analyzed by confocal microscopy using SNX2, EEA1 early endosome marker and RFP antibodies (bottom). (b) HEK293T cells were transfected for 48 h with the indicated combination of empty (Myc), Myc-tagged Vps35, SNX2 WT and SNX2Δ84. Samples were immunoprecipitated (IP) using a Myc antibody and proteins were analyzed by immunoblotting (IB) with the indicated antibodies. (c) HeLa cells were transfected two times at a 24 h interval with the indicated combination of control (CTL), SNX1 and SNX2 small interfering RNAs (siRNAs), and proteins were analyzed after 72 h by immunoblotting using the indicated antibodies. Actin was used as a loading control. (d) Mixed populations of cells transfected as in (c) were analyzed for ci-MPR, SNX2 and Vps26 localization by confocal microscopy. Arrows point to SNX1/SNX2-depleted cells. Scale bars in (a) and (d) represent 10 μm.