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. 2012 Oct 2;4(11):1186–1199. doi: 10.1002/emmm.201201438

Figure 2. Indirect immunofluorescence and Western blot analyses of TRID-mediated read-through in HEK293T cells.

Figure 2

  1. Read-through in transient transfected HEK293T cells analyzed by indirect immunofluorescence with anti-harmonin antibodies. Harmonin staining was detected in harm_a1-transfected cells whereas no staining was visible in untransfected cells and harm_a1-p.R31X controls. NB30 (2 mg/ml), NB54 (0.5 mg/ml) or PTC124 (10 µg/ml) treatment restored harmonin a1 (green) in p.R31X-transfected cells. Nuclear DNA was stained by DAPI (blue).
  2. Read-through in transiently transfected HEK293T cells analyzed by Western blot with anti-harmonin antibodies. Treatment with NB30, NB54 or PTC124 restored full-length harmonin a1 (∼80 kDa) in p.R31X-transfected cells. Actin staining (∼42 kDa) was used as loading control. For quantification of TRID-mediated read-through of the p.R31X mutation, the optical densities of harmonin a1 bands, stained by anti-harmonin antibodies, were measured and normalized to the appropriate loading control. The increase of read-through is shown as fold increase over untreated (untr.) cells. Quantitative data resulted from three to five independent repeats of the experiments, Error bars represent SD, *p < 0.05, **p < 0.01, scale bar 10 µm.