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Fig. 4 | Journal of Experimental & Clinical Cancer Research

Fig. 4

From: Nck-associated protein 1 associates with HSP90 to drive metastasis in human non-small-cell lung cancer

Fig. 4

NAP1 is stabilized by HSP90 and contributes to HSP90-mediated invasion in NSCLC cells. (a) The effect of 17-AAG (0.5–2 μM) on NAP1 protein levels in two NSCLC cell lines. (b) The effect of 17-AAG on NAP1 protein levels in the presence or absence of MG132. H661 cells were pretreated with 10 μM MG132 for 4 h and then treated with 1 μM 17-AAG for 24 h. Representative Western blotting results and quantitative data were shown in the left and right panels, respectively. (c) The effect of 17-AAG on NAP1 ubiquitination in NAP1 overexpressing H460 cells. Cells were pretreated with 10 μM MG132 for 4 h before 17-AAG treatment. Cell lysates were IP using anti-Flag antibody and immunoblotted with anti-ubiquitin antibody. β-Actin immunoblotting on total lysate is shown to normalize the input. (d) The effect of HSP90 knockdown on NAP1 protein levels in H661 cells determined by Western blotting. (e and f) The effect of HSP90 knockdown on MMP9 secretion (e) and invasion (f) in H661 cells determined by ELISA and Transwell. (g) The effect of HSP90 knockdown on the half-life of NAP1 protein determined by CHX chase assays. HSP90 knockdown H661 cells were treated with 100 μg/ml CHX for the indicated hours. (h and i) The effect of NAP1 loss on MMP9 secretion (h) and invasion (i) in HSP90 overexpressing and control H661 cells. Representative results and quantitative data were present in the left and right panels, respectively. EV: empty vector; HSP90 O/E: the HSP90 gene overexpressing vector; shCONT: non-targeting shRNA; shHSP90–1 and shHSP90–2: two shRNAs against the HSP90 gene. *p < 0.05; **p < 0.01

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