9 research outputs found
α-Actinin 1 is expressed in skeletal muscle.
<p>Light microscopy of mouse muscle culture (C2C12) at differentiation day (DD) 0 (1), DD 1 (2) and DD 3 (3) stained with antibody to α-actinin-1 (3A2).</p
WT-GNE interacts with proteins within anion-exchanged fractions of muscle cell lysate.
<p>[A] Anion exchange chromatography (0–1 M NaCl) of cell lysate (15 mg total protein content) extracted from human skeletal muscle primary culture cells. The numbers of the positive fractions appear in red on the X axis. [B] BIAcore sensorgrams of the interaction of WT-GNE with anion exchange fractions #11–16 [Resp. Diff., response difference; RU, response units; B, buffer B (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002477#s2" target="_blank">Materials and methods</a>)].</p
Kinetics BIAcore analysis for the interaction of GNE with α-actinin 1.
<p>Kinetics values of the interactions of WT and mutant GNE proteins with α-actinin 1 as calculated using 1∶1 Langmuir model.</p
LDH-B does not show BIAcore binding signal with GNE.
<p>[A] Sensorgram of the interaction of GNE with 5 µM LDH-B, compared to 5 µM α-actinin 1. [B] Sensorgram showing the effect of 2 µM LDH-B on the interaction of GNE with 0.5 µM α-actinin 1 [Resp. Diff., response difference; RU, response units].</p
LDH-B and α-actinin 1 precipitate with GNE by <i>in vitro</i> binding assay.
<p>Gelcode reagent staining of in vitro binding assay eluates resolved by SDS-PAGE. Numbers (11–13, 14–16) correspond to anion-exchange pooled fractions. A, proteins bound to GNE-bound nickel beads; B, proteins bound to GNE non-bound nickel beads; C, control proteins bound to GNE-bound nickel beads. Black arrows point to either α-actinin 1 (∼100 kDa) or to LDH-B (∼37 kDa). Both proteins were identified by MS (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002477#pone-0002477-t001" target="_blank">Table 1</a>).</p
α-Actinin is co-immunoprecipitated with FLAG-GNE in 293T-FG cells.
<p>[A] Western blot analyses with anti-FLAG antibody (A1) and anti-α-actinin antibody (A2) on 5 µg lysate-samples of FLAG-GNE (FG)-transfected (T) and untransfected (UT) 293T cells, treated with SDS sample buffer in the presence (+) or absence (−) of β-ME. [B] Co-IP results. Western blot analyses with anti-FLAG antibody and anti-α-actinin antibody of IP eluates and 5 µg lysate-samples, of FLAG-GNE (FG)-transfected (T) and untransfected (UT) 293T cells.</p
GNE and α-actinin 1 are expressed in stretched mouse muscle.
<p>Confocal microscopy of stretched mouse spinalis muscle stained with antibodies recognizing the Z line marker α-actinin-2 (ACTN2, red), α-actinin-1 (ACTN1, green), and GNE (GNE, green). α-Actinin-1 antibodies(3A2) and GNE antibodies show a distinct but overlapping staining pattern: GNE Abs recognize a diffuse band centered on the Z line while α-actinin-1 Abs stain two distinct bands on either side of the Z line. Both Abs recognize also, as a fainter staining, the sarcomeric M line. Overlap stain between α-actinin-1 and α-actinin-2 Abs is minimal.</p
MS identification of putative GNE binding partners.
<p>Bands from gradient SDS-PAGE (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002477#pone-0002477-g002" target="_blank">Figure 2</a>) were digested by trypsin and analyzed by mass spectrometry through nano-ESI-MS-MS. For each protein, the following information is indicated: protein name, molecular mass (MW), accession number in NCBInr database, percentage of protein sequence covered by the matched and sequenced tryptic fragments, number of identified peptides out of the total number of tryptic peptides, peptides sequences, mass difference between experimental and theoretical masses, and score (aa, amino acids; expt., expected; calc., calculated).</p
WT and mutant GNE proteins interact with α-actinin 1.
<p>Sensorgrams showing the interactions of GNE (WT and mutant) proteins with different α-actinin 1 concentrations and the residual curves, showing the fitness between the model and the experimental results [Resp. Diff., response difference; RU, response units].</p