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Figure 1 | Cell Communication and Signaling

Figure 1

From: Intracellular signaling prevents effective blockade of oncogenic gp130 mutants by neutralizing antibodies

Figure 1

Inducible expression of fluorescently labeled variants of WTgp130 and CAgp130. (A) T-REx-293-WTgp130-mCherry and T-REx-293-CAgp130-mCherry were left untreated or expression was induced with 20 ng/ml dox for 48 h. Cells were fixed and receptor expression was analyzed by confocal microscopy. The diagrams represent mCherry fluorescence intensities along the length of the white arrows. Scale bars: 20 μm. (B) T-REx-293-WTgp130-mCherry and T-REx-293-CAgp130-mCherry were left untreated or expression was induced with 20 ng/ml dox for 24 h. Overall receptor expression was assessed by FACS analysis of the fluorescent tag (left panel) and surface receptor expression was determined through staining with the gp130 Ab B-P8 and an APC labeled secondary Ab (right panel). Non-induced cells (filled histograms) were used as negative controls. Bar charts represent means and standard deviations from three independent experiments. Fold changes in overall and surface receptor expression as well as the ratios of surface to overall receptor expression were calculated. (C) T-REx-293-WTgp130-YFP and T-REx-293-CAgp130-YFP were left untreated or expression was induced with 20 ng/ml dox for the indicated periods of time. TCLs were analyzed by immunoblotting using an Ab raised against a C-terminal peptide of gp130 and an actin Ab as loading control. (D) T-REx-293-WTgp130-YFP and T-REx-293-CAgp130-YFP were incubated with 20 ng/ml dox for 24 h. TCLs were left untreated or were subjected to endoH digestion. Subsequently, lysates were analyzed by immunoblotting using Abs against GFP/YFP and actin as loading control.

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