Prohibitin: A Novel Molecular Player in KDEL Receptor Signalling

Figure 3

Effects of PHB knock-down on five proteins localised at the ER–Golgi interface. (a) Analysis of PHB knockdown efficiency. HeLa-myc cells were treated with siRNAs against PHB (PHB siRNA) for the indicated times. Scrambled interfered cells (Control) are shown as reference. The cells were lysed, and the proteins were analysed by Western blotting for PHB expression levels. Actin was used as the loading control. (b) PHB knockdown affects the intracellular distribution of ER and Golgi proteins. Mock-interfered (Control) and PHB-interfered (PHB siRNA) COS-7 cells were fixed and stained for GM130, p115, β subunit of the COPI coatomer complex (β-COP), GBF1 and SEC31, as indicated. The images are representative of two independent experiments. Scale bars, 10 μm. (c) Quantification of β-COP immunofluorescence levels on the Golgi complex. Data are means ± SEM of β-COP immunofluorescence from two independent experiments, with at least 25 cells quantified per experiment. compared to control cells (-test). AU: arbitrary units. (d) PHB knockdown does not affect the expression levels of ER and Golgi proteins. COS-7 cells treated as in B were lysed and their proteins analysed by Western blotting using antibodies to GM130, p115, β-COP, GBF1 and SEC31. The levels of PHB knockdown were investigated as a control.

(a)

(b)

(c)

(d)