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. 2020 Mar 11;21(5):e48901. doi: 10.15252/embr.201948901

Figure EV5. Removal of cholesterol from lysosomal compartment does not recover impaired Rab8 trafficking and its activation in TMEM135‐depleted cells.

Figure EV5

  1. RPE1 cells were transfected with siRNAs as indicated and immunostained with antibodies against Rab8 (red) and γ‐tubulin (green). Scale bar, 10 μm. Representative magnified images are shown from cells labeled with white asterisks.
  2. Quantification of the percentage of cells with Rab8 localized at the centrioles. Data represent mean ± SD (n = 3 experiments), and 200 cells were scored per condition per experiment; *P < 0.05, Student's t‐test.
  3. (Upper panel) Cells were transfected as indicated, and cell lysate was incubated with purified GST‐JCF1 (RBD) fusion protein. The amount of GTP‐Rab8 bound to GFT‐JCF1(RBD) was analyzed by Western blotting with Rab8 antibody. (Lower panel) The intensity of bands was quantified by ImageJ software. The amount of GTP‐Rab8 was normalized to the control level. The bar graph represents mean ± SD (n = 3 experiments). *P < 0.05, Student's t‐test.

Source data are available online for this figure.