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. 2017 Dec 27;2017:8607532. doi: 10.1155/2017/8607532

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Copyright © 2017 Xin Xing et al.

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Characterization of the SMC domain of Nesprin-2. (a) Schematic of Nesprin-2 (not drawn to scale). The location of the SMC domain (spectrin repeats 11–13) and the C-terminal spectrin repeats (53–56) is shown. Epitopes of antibodies used are indicated above the schematic. ABD, actin binding domain; ovals, spectrin repeats. The spectrin repeat domain starts at position 308. (b) Sequence comparison of the Nesprin-2-SMC domain with coiled-coil regions of SMC2 and SMC4. The sequence comparison was performed using LALIGN, the Pairwise Sequence Alignment tool from EMBL-EBI (https://www.ebi.ac.uk/Tools/psa/lalign/). Nesprin-2 (NCBI GenBank accession number AF435011.1), SMC2 (NCBI GenBank accession number O95347.2), and SMC4 (NCBI GenBank accession number Q8WXH0.3) were used. :, identical amino acid; ., conservative substitution. (c) Analysis of Nesprin-2-SMC by gel filtration chromatography. UV traces of the elution profile are shown. Nesprin-2 SMC (calculated molecular weight 39 kDa). Molecular weight markers were ovalbumin (43 kDa), conalbumin (75 kDa), and aldolase (158 kDa). (d) Analysis of chemically crosslinked Nesprin-2-SMC. Zero-length cross-linking reagent EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride) was used at decreasing concentrations. The proteins were separated by SDS-PAGE (10% acrylamide) and stained with Coomassie Blue. (e) Schematic representation of Myc-tagged Nesprin-2-SMC polypeptides. Amino acid positions refer to human Nesprin-2 giant (accession number AF435011.1). (f) Interaction of GST-Nesprin-2-SMC with individual Myc-tagged spectrin repeats derived from Nesprin-2-SMC and expressed in COS7 cells. GST-Nesprin-2-SMC was used for pulldown (right panel). Western blots were probed with mAb 9E10 specific for Myc. Asterisk, endogenous Myc [25]. (g) Specificity of the Nesprin-2-SMC interaction. Myc-SR53–56 expressed in COS7 cells was used for pulldowns with GST for control and GST-Nesprin-2-SMC. COS7 and COS7 Myc-SR53–56 represent whole cell lysates. The Ponceau S stained blot and the corresponding blot probed with mAb 9E10 are shown. MW, molecular weight marker (from top to bottom: 200, 130, 100, 70, 55, 35, and 25 kDa).