Fig. 8. UV-induced EGFR internalisation was independent of the receptor tyrosine kinase activity. (Upper panels) Cells were either left untreated, stimulated with 5 nM EGF for 10 minutes, with or without preincubation in 100 nM PD153035 for 2 hours. Cell lysates were subject to SDS-PAGE and western immunoblotting with anti-pY1173 (left) or anti-EGFR (right). The specific EGFR tyrosine kinase inhibitor PD153035 effectively inhibited the EGF-mediated receptor tyrosine phosphorylation (left). Whereas pretreatment with PD153035 did not inhibit the UV-induced EGFR gel mobility shift, the mobility shift induced by EGF was inhibited (right). C and E denote control and EGF-stimulated cells, respectively. (Lower panel) Cells were either left untreated (A), stimulated with 5 nM EGF for 10 minutes (B), with pretreatment with PD153035 (C), or pretreated with PD153035 followed by exposure to 200 J/m² UV, and incubated for 10 minutes (D). Immunofluorescense confocal microscopy with anti-EGFR demonstrated an EGF-induced redistribution of EGFR from the plasma membrane (A) to vesicles (B). The EGF-mediated receptor internalisation was inhibited by pretreatment with 100 nM PD153035 for 2 hours (C). Inhibition of the UV-induced EGFR internalisation was not observed with pretreatment with PD153035 (D). Bar, 10 µM.