Fig. 1.

Gadkin modulates endosomal recycling. (A) Perinuclear AP-1 is reduced in Gadkin knockdown cells. Sum fluorescence intensity of AP-1 within the TGN46-positive area of control or Gadkin siRNA-treated (B) HeLa cells. (C) Knockdown of Gadkin facilitates Tf recycling. siRNA-transfected HeLa cells were analyzed for their ability to recycle internalized ¹²⁵I-Tf. Data represent mean values ± SEM (n = 11 values for each data point; five independent experiments; *, P < 0.05; ***, P < 0.001; Student's t test). Knockdown was verified by immunoblot analysis (Inset). (D) Effect of Gadkin WT or mutant expression in cells depleted of endogenous Gadkin on Tf recycling. siRNA- and DNA-transfected cells were allowed to internalize Alexa⁵⁶⁸-labeled Tf, followed by a 30 min chase. Remaining Tf was quantified by fluorescence microscopy. Bars represent mean values ± SEM (n = 20–25 cells for each sample; ***, P < 0.001; one-way ANOVA followed by Tukey's Multi Comparison Test). (Inset) Gadkin-eGFP expression in Gadkin-knockdown HeLa cells. (E, Left) Total N-cadherin levels in Gadkin-depleted Cos7 cells. (E, Right) Surface levels of N-cadherin in Gadkin-depleted Cos7 cells assayed by surface biotinylation. Signals were quantified with ImageJ. Immunoblot analysis of samples representative of at least three independent experiments is shown below the bar diagram.