Figure 5
The knockdown of Hsp70 negatively affects surface-localized alpha-enolase. (a) HB2, MCF-7 and MCF-7R cells were treated with Hsp70-specific siRNA (+) or unrelated siRNA as a control (−), and immunoblotting analysis of total lysates with anti-Hsp70 and anti-alpha-enolase (α-Eno) was performed. β-actin is shown as a control of the total proteins loaded per lane. (b) Quantification of Hsp70 and alpha-enolase in Hsp70-silenced cells. Data were normalised to the densitometric signals of β-actin and expressed relative to the level detected in cells treated with unrelated siRNA (siControl), set at 1. (c,d) Live on-cell western of Hsp70-silenced cells. The bar graph in (c) shows the relative mean infrared intensity for membrane Hsp70, corrected for nuclei staining. Transiently-silenced cells were either untreated or treated with 5 µg/ml LPS or 0.1 µg/ml EGF for 24 hours, and then analysed for the expression of surface-localized alpha-enolase by on-cell western (d). Results are the average of three independent experiments, error bars represent standard deviation and p values (*P < 0.05, **P < 0.01, ***P < 0.001) indicate statistical significance relative to the untreated control.
