anti cap3  (Cell Signaling Technology Inc)

Journal: Journal of biomedical science

Article Title: Similar dose-dependence of motor neuron cell death caused by wild type human TDP-43 and mutants with ALS-associated amino acid substitutions.

doi: 10.1186/1423-0127-20-33

Figure Lengend Snippet: Figure 2 Apoptotic assays of transfected NSC34 and Neuro2a cells. (A) Comparison of apoptotic death induced by exogenous WT and MT hTDP-43. Cell death was assayed by measurement of the activities of caspase 3/ 7 at 24 hr and 72 hr post-transfection with the expression plasmids (5 ug/ 106 cells). Mock, cells without transfection; C, cells transfected with the pEF vector; Stau. 5 uM, cells treated with 5 uM of staurosporine for 6 hr to induce apoptosis. The folds of the caspase activities relative to that of the Mock sample were calculated and shown. Note the significant increase of the caspase 3/ 7 activities in NSC34 cells (left panel), but not Neuro2a cells (right panel), induced by the two MT hTDP-43 forms at 72 hr post-transfection. (B) Comparison of plasmid dose-dependent apoptotic death induced by exogenous WT and MT hTDP- 43. Apoptotic cell death was determined by immunofluorescence staining of cleaved caspase 3. The extents of apoptotic cell death of NSC34 cells and Neuro2a cells at 72 hr post-transfection with different amounts of the expression plasmids were assayed by immunofluorescence staining with the antibodies 2E2D3 and Ac-cap 3. Means of three independent experiments (S.D.) are plotted in the upper 2 panels, with the % of hTDP-43-positive cells that are also Ac-cap 3-positive as a function of the doses of transfection. Approximately 1% of cells transfected with the pEF vector were Ac-cap3 positive (◆in the two plots). Representative photographs are shown below the plots, with the apoptotic nuclei/ cleaved caspase 3-positive cells indicated by the arrowheads. For both the NSC34 and Neuro2a sets, two Ac-cap 3-positive cells (the boxed areas) are magnified for better visualization. Scale bar, 10 μm. *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: The commercial antibodies used in this study included a rabbit anti-TDP-43 polyclonal antibody (pAb) raised against a.a 1–260 of human TDP-43 and recognizing human as well as mouse TDP-43 (Gene Tex), a human specific mouse monoclonal antibody (mAb) against the same TDP-43 sequence (2E2-D3) (Abnova), anti-Myc mAb (LTK), anti-α-tubulin (Sigma), anti-cleaved caspase 3 (Asp175) (Ac-cap3) (Cell Signaling), and anti-Hsp70 (Chmicon).

Techniques: Transfection, Comparison, Expressing, Plasmid Preparation, Immunofluorescence, Staining