Fig. 1

SorLA interacts with PICK1. A Principle of the mammalian-2-hybrid system. DBD, DNA-binding domain; Luc, firefly luciferase; Pol, polymerase; TAD, transcription activation domain; UAS, upstream activating sequence. B Primary sequence of the SorLA cytoplasmic domain (CD) (top) and of the Sortilin cytoplasmic domain. Protein interaction motifs mutated in (C) are underlined. C Mammalian-2-Hybrid analysis of PICK1 with the cytoplasmic domain of Sortilin or SorLA. Co-transfections of N2a cells with fusion constructs of the GAL4 DNA binding domain with wild-type Sortilin or SorLA or the mutant cytoplasmic domain of SorLA and a fusion construct of the VP16 transcriptional activation domain with full length PICK1 were analyzed. Terminal or mutated amino acid sequences of the cytoplasmic domains are indicated on the left. Motifs marked in (B) are indicated with the same color code. Luciferase activity was normalized to the fluorescence generated by a co-transfected eGFP vector. Relative luciferase activity (mean ± SD) based on a typical experiment performed in septuplicate. D Detection of PICK1-Myc in stably transfected CHO-cells by immunoblotting using an anti-Myc or anti-PICK1 antibody (left). Pull-down of PICK1-Myc from stably transfected CHO-cells (right). Precipitation was performed with GST or GST fused to the cytoplasmic domain of SorLA and analyzed by immunoblotting using an anti-Pick1 antibody. E Pull down of PICK1 from murine brain lysates. GST or GST fused to the cytoplasmic domain of SorLA was used to pull down from brain lysates and analyzed by immunoblotting using an anti-PICK1 antibody