Expression in mouse spinal cord.

<p>IF images of the expression of eEF1A2 and eEF1Bα (top panel) or eEF1Bδ (bottom panel) on mouse spinal cord.</p

Viability and cell cycle distribution of cells after ablation of eEF1B subunits.

<p>Panel A: A decrease in cellular metabolism is observed when any of the eEF1B subunit protein level is decreased by siRNAs in HeLa, HTC116 and DLD1 cells. Cell metabolism was assessed by the Alamar blue assay. Data were obtained from the mean of three or more independent experiments in HeLa, HCT116 and DLD1 cells, with more than 10 wells each. Error bars indicate +- SEM; n of wells>10; n = 3−4; *, P<0.05; ***, P<0.001 from non-targeting siRNA. Panel B: Knockdown of eEF1B subunits leads to altered cell cycle profile in three cell lines: representative images of the flow cytometry analysis. Error bars indicate +- SEM; n = 3; *, P<0.05; **, P<0.01 of non-targeting siRNA.</p

Proximity ligation assays for eEF1A and eEF1B.

<p>Panel I: PLA on HeLa cells. A. Negative control with both primary antibodies omitted. B. eEF1A2 antibody only. C. PLA of eEF1A2 and eEF1Bα. D. PLA of eEF1A2 and eEF1Bδ. E. PLA of eEF1A2 and eEF1Bγ. F. PLA of eEF1A2 and TK1 as negative control. Images in the squares are higher magnification of selected areas. Panel II: PLA on NIH-3T3 cells stably expressing V5-tagged eEF1A1. A. Negative control with both primary antibodies omitted. B. V5 antibody only. C. PLA of eEF1Bα and V5. D. PLA of eEF1Bα and TK1. E. PLA of eEF1Bδ and V5. F. PLA of eEF1Bδ and TK1. G. PLA of eEF1Bγ and V5. H. PLA of eEF1Bγ and TK1. Images in the squares are higher magnification of selected areas. Panel III: PLA on NIH-3T3 cells stably expressing V5-tagged eEF1A2. A. Negative control with both primary antibodies omitted. B. V5 antibody only. C. PLA of eEF1Bα and V5. D. PLA of eEF1Bα and TK1. E. PLA of eEF1Bδ and V5. F. PLA of eEF1Bδ and TK1. G. PLA of eEF1Bγ and V5. H. PLA of eEF1Bγ and TK1. Images in the squares are higher magnification of selected areas.</p

Expression analysis of eEF1B subunits in cell lines and tissues.

<p>Panel A: Immunoblot of eEF1Bα, eEF1Bδ and eEF1Bγ protein expression in cell lines. GAPDH was used as a loading control. Panel B: Immunoblot of eEF1B subunits in 24-day old mice. Each tissue is shown twice, the left hand sample is from a wild type mouse and the right hand sample from a wasted homozygous mouse. SC =  spinal cord. The second row shows a longer exposure of the eEF1Bδ blot, revealing the expression of the longer isoform. GAPDH was used as a loading control. Panel C: Immunoblot of eEF1Bδ in an extended panel of mouse tissues showing brain and testis-expression of the longer isoform (top panel shows a longer exposure). GAPDH was used as a loading control. Panel D: Immunoblot of eEF1B subunits expression in brain (Br) and liver (Li) throughout late embryonic and postnatal mouse development. GAPDH was used as a loading control.</p

Immunohistochemistry of eEF1B subunits in human and mouse tissues.

<p>Immunohistochemistry of eEF1B subunits in human and mouse brain and pancreas. Proteins detected through primary antibody incubation, HRP mouse + rabbit secondary antibody and subsequent incubation with DAB. Positive signal is indicated by the presence of brown DAB reaction product. Panels show eEF1Bα (<b>b</b> and <b>f</b>), eEF1Bδ(<b>c</b> and <b>g</b>) and eEF1Bγ (<b>d</b> and <b>h</b>) Incubation with secondary antibody only was used as negative control (<b>a</b> and <b>e</b>). Bar (top left micrograph) represents 100 and 50 µm respectively.</p

Using RNA interference to knock down expression of eEF1B subunits.

<p>Immunoblots of protein extracts from cell lines after RNA interference. Panel A: eEF1Bα, eEF1Bδ and eEF1Bγ protein level efficiently knocked down by three different siRNAs in HeLa cells 72 h after transfection. GAPDH was used as a loading control. Panel B: eEF1Bα, eEF1Bδ and eEF1Bγ protein level efficiently knocked down by three different siRNAs in HCT116 and DLD1 cells 72 h after transfection. GAPDH was used as a loading control.</p

Grip strength analysis of aged mice.

<p>Grip strength data for the entire ageing study. Graphs on the left display male data and graphs on the right display female data. Top graphs display forelimb data only and the bottom 2 graphs display data from all four limbs. WT indicates wild-type animals, HET indicates heterozygote animals. Error bars represent the standard error of the mean.</p

Immunohistochemistry in aged spinal cord sections.

<p>Expression of phosphorylated neurofilaments, GFAP, and eEF1A2 in cervical spinal cord sections from 21 month old mice. The top panel in each case shows a section with primary antibody omitted from the protocol, the second panel from the top shows sections from a 24 day old wasted homozygote as a control, and the bottom two panels show sections from an aged matched wild-type and heterozygous male. The NF staining clearly shows perikaryal accumulation of NF staining in the wasted mouse section but not in the aged <i>+/wst</i> mouse. The GFAP staining shows a characteristic pattern of reactive astrocytes throughout the grey matter of the spinal cord, even in the aged wild-type mouse. The eEF1A2 shows no staining at all in the section from the <i>wst/wst</i> mouse as expected, and fainter but easily detectable, albeit reduced, staining in the aged <i>+/wst</i> sample.</p

Grip strength analysis of young wasted mice.

<p>Forelimb (top panel) and all four limbs (bottom panel) grip strength analysis of wasted mice. The daily grip strength reading of 3 tests (measured in Newtons) were normalised to body weight (measured in grams). P values were calculated comparing wasted mice with controls (+/+ and <i>+/wst</i> combined). * indicates a P value<0.05, ** indicates a P value<0.01, and *** indicates a P value <0.001.</p

Protein expression in aged mice.

<p>Western blots showing protein expression of eEF1A2 and GAPDH (as a loading control) of animals from the ageing cohort. Three 21month old animals from each group are shown. WT indicates wild-type animals, HET indicates heterozygous animals, M indicates a muscle sample (as an eEF1A2 positive control) and L indicates a liver sample (an eEF1A2 negative control), both from wild-type mice.</p