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. 2014 Aug 1;3(9):803–814. doi: 10.1242/bio.20148771

Fig. 5. Rab35 and MICAL-L1 recruit Rab36 to recycling endosomes.

Fig. 5.

(A) Accumulation of Rab35 and Rab36 in the perinuclear area of PC12 cells in response to NGF stimulation. After NGF stimulation for 0 hr, 1 hr, and 6 hr, PC12 cells were fixed and stained with anti-Rab35 antibody, anti-Rab36 antibody, and DAPI. (B) Colocalization between MICAL-L1 and Rab36 in PC12 cells. After NGF stimulation for 6 hr PC12 cells were fixed and stained with anti-MICAL-L1 antibody, anti-Rab36 antibody, and DAPI. The insets in panels A and B are magnified views of the boxed areas. (C,D) Intensity scatter plot of Rab35 signals versus Rab36 signals (C) and of MICAL-L1 signals versus Rab36 signals (D) in PC12 cells after NGF stimulation for 6 hr. The Pearson's correlation coefficient (PCC) value (mean ± SD) for the relation between them is shown at the bottom (n = 30 from 3 independent experiments). (E) Unaltered expression of Rab36 in PC12 cells during NGF stimulation. After NGF stimulation for 0 hr, 1 hr, and 6 hr, cell lysates of PC12 cells were immunoblotted with anti-Rab36 antibody and anti-actin antibody. (F) Disappearance of Rab36 signals from the perinuclear area of Rab35-depleted and MICAL-L1-depleted PC12 cells. After NGF stimulation for 6 hr PC12 cells treated with siControl, siRab35, or siMICAL-L1 were fixed and stained with anti-Rab36 antibody and DAPI. (G) Perinuclear Rab36 signals (mean and SE) of siControl-treated, siRab35-treated, and siMICAL-L1-treated PC12 cells after NGF stimulation for 6 hr (n = 60 from 3 independent experiments). (H,I) Unaltered expression of Rab36 in Rab35-depleted PC12 cells (H) and MICAL-L1-depleted PC12 cells (I). Cell lysates of PC12 cells treated with siControl, siRab35, or siMICAL-L1 were immunoblotted with anti-Rab36 antibody and anti-actin antibody. (J,K) Translocation of Rab36 to the plasma membrane after expression of Rab35 and MICAL-L1 at the plasma membrane. After NGF stimulation for 6 hr PC12 cells transiently expressing mStr–Rab35-Q67L–CAAX and EGFP–Rab36-Q116L together with Myc–MICAL-L1-CC (J) or Myc–MICAL-L1-ΔCC (K) were fixed and stained with anti-Myc tag antibody and DAPI. Fluorescence intensity along the broken arrows is shown at the bottom. Scale bars: 10 µm.