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. 2014 Dec;184(12):3262–3271. doi: 10.1016/j.ajpath.2014.08.014

Figure 5.

Figure 5

FIH-1/LRRK1 regulates EGFR signaling in part by interfering with endosomal trafficking. HEKs (A and B) and limbal-derived corneal epithelial hTCEpi cells (C and D) were transfected with siLRRK1 (A and C), siFIH-1 (B and D), or siCTRL. After transfection (72 hours), cells were treated with 20-μmol/L chloroquine (Chlo) for 2 hours or vehicle control (water), and lysates were harvested and immunoblot-analyzed with antibodies against FIH-1, LRRK1, p-EGFR (Y1045 or Y1068), and GAPDH. E and F: hTECpi cells transduced with an empty vector (LZRS) or FIH-1 were treated with fluorescence-labeled EGF (Alexa Fluor 647 EGF complex; red). Cells were harvested at 30 minutes after treatment and stained for EEA1 (an early endosome marker; green). Colocalization of EGF and EEA1 were highlighted as white pixels (E) and Pearson's coefficient was analyzed by ImageJ (F). The box-and-whiskers plots represent the median line; whiskers, minimum to maximum. n = 5 (F). ∗P < 0.05.