Prohibitin: A Novel Molecular Player in KDEL Receptor Signalling

Figure 4

PHB knock-down hinders KDELR-dependent SFKs activation. (a) Depletion of PHB inhibits SFKs activation. HeLa-myc cells were treated with scrambled siRNAs (Control) or siRNAs against PHB (PHB siRNA) for 96 h. After infection with VSV for 45 min, the cells were incubated at 40°C for 3 h (temperature block) and then shifted to 32°C for 30 min (block release). The cells were lysed and analysed by Western blotting for active phosphorylated SFKs (p-SFKs). The total SFKs (SFKs) was used as the loading control, while the knock-down levels were assessed with a PHB antibody. The images shown are representative of two independent experiments. (b) Depletion of PHB inhibits SFKs activation on the Golgi complex. HeLa-myc cells were treated as in (a). Following the 30 min of temperature-block release, the control cells and siRNA-treated cells were fixed and stained for GM130 (marker for Golgi area definition; green) and for active SFKs (p-SFKs; red); the merged images are also shown. Scale bars, 10 μm. (c) Quantification of p-SFKs immunofluorescence intensity on the Golgi complex of HeLa-myc cells treated as in (a). Data are means ± SEM of three independent experiments, with at least 50 cells quantified per experiment. compared to control cells at 32°C (-test).

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