Fig. 4.

In vivo investigation of the dimer interface. (A) Rubicon mutant expression. HeLa cells were transfected with plasmids encoding eight FLAG-tagged mutants and wild-type Rubicon and lysed after 24 h. Expression levels of Rubicon were detected by Western blotting with anti-FLAG antibody. (B and C) Colocalization of Rubicon mutants with Rab7. HeLa cells were cotransfected with FLAG-tagged Rubicon mutants and mRFP–Rab7 and stained with GFP–anti-FLAG antibody after 48 h. (B) Rubicon, Rab7, and merged images from boxed areas are magnified and shown from left to right, respectively. White arrows indicate colocalized signals. (Scale bars, 20 μm.) (C) Quantification of colocalization between Rubicon mutants and Rab7. Colocalization was analyzed in ImageJ and is shown via Pearson’s R value. Plot shows mean values +/− SD (n = 3). (D and E) Effect of Rubicon variant overexpression on autophagy. HeLa cells stably expressing tfLC3 (mRFP–EGFP–LC3) were transfected with each FLAG–Rubicon variant plasmid and stained with anti-FLAG antibody. (D) Cells were incubated in starvation medium. Cells expressing FLAG–Rubicon are indicated with asterisks. (Scale bars, 20 μm.) (E) Signal intensities of EGFP and mRFP were quantified using ImageJ and are shown as EGFP/mRFP ratio normalized to the value from vector-transfected cells. * indicates P < 0.05, ** indicates P < 0.01; ns indicates P > 0.05.