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. 2012 Jul 23;7(7):e41537. doi: 10.1371/journal.pone.0041537

Figure 1. Ablation of NEGR1 in mouse.

Figure 1

A. Generation of Negr1 knockout mice. Schematic diagram of the targeting vector, the wild-type and mutant alleles. Only relevant restriction sites are shown. The positions of the external southern blot probe, as well as the PCR primers, are indicated by asterisks and arrows, respectively. B. Genomic southern blot using the restriction enzymes EcoRI and SpeI. Bands resulting from introduction of a new EcoRI site and deletion of a SpeI site are indicated by asterisks. C. PCR genotyping of transgenic mice wild-type (+/+), heterozygous (+/−) and homozygous (−/−) mice. D. Western blot probed with antibodies specific to NEGR1 and β-actin showing complete loss of NEGR1 protein in knockout (−/−) mice. E. Sequencing of genomic DNA reveals a T-A mutation that converts an isoleucine residue to asparagine at position 87 (I87N). F. Western blots of brain lysate from Negr1-I87N mice probed with an anti-NEGR1 antibody. G. NEGR1-immunoblotting of NSC-34 cell lysates overexpressing NEGR1-WT, NEGR1-I87N, and mock-transfected cells. H–I. The Negr1-I87N mutation causes ER retention of NEGR1. Confocal images showing NSC-34 cells co-expressing NEGR1-WT (H) or NEGR1-I87N (I) together with DsRed-ER. NEGR1-WT is predominantly localized at the plasma membrane whereas distribution of NEGR1-I87N clearly overlaps with the DsRed-fluorophore-labeled ER. Nuclei are visualised by DAPI. Scale: 10 µm.