Journal: International Journal of Cell Biology

Article Title: Cytoskeletal Interactions at the Nuclear Envelope Mediated by Nesprins

doi: 10.1155/2012/736524

Figure Lengend Snippet: Nesprin-1 N-terminal sequences interact with Nesprin-3. (a) COS7 cell lysates expressing the N-terminal Nesprin-3 (GFP-Nesprin-3-ΔC) fusion protein were incubated with the indicated Nesprin-1-165 polypeptides fused to GST. GST was used as control. The cell lysates (input and supernatant, upper and middle panel) and the pelleted protein (lower panel) were subjected to SDS-PAGE followed by western blotting using mAb K3-184. P, pellet; S, supernatant. (b) Yeast-two hybrid analysis of the interaction between Nesprin-1 and Nesprin-3. Fragments corresponding to Nesprin-1-165 full length and the indicated Nesprin-1-165 polypeptides were fused to the Gal4 DNA binding domain (BD), the N-terminal part of Nesprin-3 lacking the KASH domain (Nesprin-3-ΔC) was fused to the GAL4 activation domain (AD). The corresponding plasmid pairs were cotransformed into yeast strain Y190 and interaction was assessed by filter lift β -galactosidase assay. Blue colour development indicates β -galactosidase activity.

Article Snippet: COS7 cells were transiently transfected with Nesprin-1-165-GFP (aa 1–1431) [ ], GFP-1-286 (ABD), GFP-573-858, GFP-859-1144, GFP-1145-1431, GFP-Nesprin-3-ΔC (aa 1–437; cloned into the AhdI/SacII site of the EGFP-C2 vector), and HA-Nesprin-3 (aa 1−975; cloned into KpnI/NotI digested pCMV-HA) by electroporation (Gene Pulser Xcell, Bio-Rad).

Techniques: Expressing, Incubation, SDS Page, Western Blot, Binding Assay, Activation Assay, Plasmid Preparation, Activity Assay

Journal: International Journal of Cell Biology

Article Title: Cytoskeletal Interactions at the Nuclear Envelope Mediated by Nesprins

doi: 10.1155/2012/736524

Figure Lengend Snippet: F-actin interaction of Nesprin-1 ABD and Nesprin-3. (a) Nesprin-1 ABD binds to F-actin in vitro in the presence of Nesprin-3 in an F-actin co-sedimentation assay. Recombinantly expressed and purified GST-Nesprin-3 SR1,2,3 (GST NE3 SR1,2,3), and Nesprin-1 ABD (ABD NE1) were precleared and incubated with and without actin at room temperature under polymerizing conditions. High-speed centrifugation at 100,000 ×g for 1 h at 4°C was followed by separation of the protein mixture into supernatant (S) and pellet (P) fractions. ABD NE1 and GST NE3 SR1,2,3 bound to F-actin and were observed in the pellet fraction. F-actin binding was also observed when both proteins were added to the polymerization assay (actin + ABD NE1 + GST NE3 SR1,2,3). Samples were treated with 5x SDS-sample buffer and proteins separated on SDS-PA gels (12% acrylamide) and stained with Coomassie Brilliant Blue. The two closely migrating polypeptides in the Nesprin-1 ABD sample were identified as Nesprin-1 ABD by mass spectrometry. Arrows from top to bottom indicate the location of GST NE3 SR1,2,3, actin, and ABD NE 1. (b) Spectrin repeats of Nesprin-3 bind to F-actin. Nesprin-3 spectrin repeats SR1,2, SR2, and SR1 were used in the assay. Only SR1,2 (26.7 kDa) copelleted with F-actin (40 kDa) whereas SR2 (14.6 kDa) and SR1 (12.1 kDa) stayed in the supernatant. Proteins were separated on SDS-PA gels (18% acrylamide). The fast migrating protein in the SR1 sample corresponds also to SR1.

Article Snippet: COS7 cells were transiently transfected with Nesprin-1-165-GFP (aa 1–1431) [ ], GFP-1-286 (ABD), GFP-573-858, GFP-859-1144, GFP-1145-1431, GFP-Nesprin-3-ΔC (aa 1–437; cloned into the AhdI/SacII site of the EGFP-C2 vector), and HA-Nesprin-3 (aa 1−975; cloned into KpnI/NotI digested pCMV-HA) by electroporation (Gene Pulser Xcell, Bio-Rad).

Techniques: In Vitro, Sedimentation, Purification, Incubation, Centrifugation, Binding Assay, Polymerization Assay, Staining, Mass Spectrometry

Journal: International Journal of Cell Biology

Article Title: Cytoskeletal Interactions at the Nuclear Envelope Mediated by Nesprins

doi: 10.1155/2012/736524

Figure Lengend Snippet: Nesprin-3 recruits intermediate filaments to the nuclear envelope in COS7 cells. COS7 cells stained for vimentin reveal the typical cytoskeletal staining (A-A′′). In HA-Nesprin-3 transfected COS7 cells vimentin was recruited to the NE and co-localized with HA-Nesprin-3 (B-B′′). Confocal images are shown. Size bar, 10 μ m.

Article Snippet: COS7 cells were transiently transfected with Nesprin-1-165-GFP (aa 1–1431) [ ], GFP-1-286 (ABD), GFP-573-858, GFP-859-1144, GFP-1145-1431, GFP-Nesprin-3-ΔC (aa 1–437; cloned into the AhdI/SacII site of the EGFP-C2 vector), and HA-Nesprin-3 (aa 1−975; cloned into KpnI/NotI digested pCMV-HA) by electroporation (Gene Pulser Xcell, Bio-Rad).

Techniques: Staining, Transfection

Journal: International Journal of Cell Biology

Article Title: Cytoskeletal Interactions at the Nuclear Envelope Mediated by Nesprins

doi: 10.1155/2012/736524

Figure Lengend Snippet: Illustration of Nesprin interactions at the outer nuclear membrane surface. The inner (INM) and outer (ONM) nuclear membranes are shown to allow simultaneous documentation of the interaction of Nesprins with SUN-domain complexes. Nesprins are aligned along the nuclear envelope. Nesprin-1/-2 ABDs interact with F-actin, N-terminal Nesprin-1 spectrin repeats interact with Nesprin-3, the latter can also interact with the ABD of plectin. Therefore, Nesprins integrate the NE with the major cytoskeletal filaments allowing the formation of a multifunctional Nesprin network along the surface of the ONM. INM located Nesprins have been omitted from this scheme. PNS, perinuclear space.

Article Snippet: COS7 cells were transiently transfected with Nesprin-1-165-GFP (aa 1–1431) [ ], GFP-1-286 (ABD), GFP-573-858, GFP-859-1144, GFP-1145-1431, GFP-Nesprin-3-ΔC (aa 1–437; cloned into the AhdI/SacII site of the EGFP-C2 vector), and HA-Nesprin-3 (aa 1−975; cloned into KpnI/NotI digested pCMV-HA) by electroporation (Gene Pulser Xcell, Bio-Rad).

Techniques:

Journal:

Article Title: Isolation and Characterization of a Histidine Biosynthetic Gene in Arabidopsis Encoding a Polypeptide with Two Separate Domains for Phosphoribosyl-ATP Pyrophosphohydrolase and Phosphoribosyl-AMP Cyclohydrolase

doi:

Figure Lengend Snippet: Summary of the E. coli strains, plasmids, and oligonucleotides used in this study

Article Snippet: These recombinant HIS1 and hisA proteins were produced in E. coli as fusion proteins with the maltose-binding protein using a pMAL-c2 vector (New England Biolabs) and affinity purified using amylose resin columns (New England Biolabs).

Techniques: Plasmid Preparation

Journal:

Article Title: Isolation and Characterization of a Histidine Biosynthetic Gene in Arabidopsis Encoding a Polypeptide with Two Separate Domains for Phosphoribosyl-ATP Pyrophosphohydrolase and Phosphoribosyl-AMP Cyclohydrolase

doi:

Figure Lengend Snippet: At-IE-dependent AICAR production determined in the assay mixture containing S. cerevisiae HIS1, the hisA protein of E. coli, and one of the expressed recombinant proteins. The At-IE protein without the putative chloroplast transit peptide, the putative PRA-PH (hisE), and the putative PRA-CH (hisI) domains were expressed as the fusion proteins with a maltose-binding protein using a pMAL-c2 bacterial expression vector. The expression vectors used were the same as those presented in Figure ​Figure77.

Article Snippet: These recombinant HIS1 and hisA proteins were produced in E. coli as fusion proteins with the maltose-binding protein using a pMAL-c2 vector (New England Biolabs) and affinity purified using amylose resin columns (New England Biolabs).

Techniques: Recombinant, Binding Assay, Expressing, Plasmid Preparation