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. Author manuscript; available in PMC: 2009 Feb 3.

Published in final edited form as: J Cell Sci. 2008 Apr 22;121(Pt 10):1649–1660. doi: 10.1242/jcs.025726

Aberrant conjugation of Atg5 with Atg12 after Rab5 inhibition. a. HeLa cells transfected with control or Atg7 siRNA for 48h were subjected to second round of transfection with GFP-Atg5 together with the siRNA for another 24h. Then the cells were fixed following saponin extraction to visualise the GFP-Atg5 structures (green). b. HeLa cells transfected with control vector or DN-Rab5 together with HA-Atg12 and Atg5 were subjected to western blot analysis with anti-HA antibody to detect free Atg12 and the Atg5-Atg12 complex. The experiment assays the proportion of transfected Atg12 that gets conjugated to Atg5 by assessing the ratio of Atg5-Atg12 complex to free Atg12 as shown in the graph. Atg5-Atg12/Atg12 ratio from 4 independent experiments is shown *** - P < 0.0001. c. Similar experiments to Fig. 3b were performed in the presence or absence (control) of 3-methyladenine (3-MA; left) or with control or Vps34 siRNA (right). Atg5-Atg12/Atg12 ratio from 3 (for Vps34 siRNA) and 4 (for 3MA) independent experiments is shown. *** - P < 0.0001, ** - P < 0.001. d. HeLa cells transfected simultaneously with Rab5a, Rab5b and Rab5c siRNA (Rab5; left), Atg7 (right) or control siRNA for 48h were subjected to second round of transfection with Atg5 and HA-Atg12 together with the siRNA for another 24h. Western blot analysis was performed with anti-HA antibody to detect free Atg12 and the Atg5-Atg12 complex. Representative images from two independent, reproducible experiments are shown. e. Immunostaining of HeLa cells transfected with control (with or without 3-MA) or DN-Rab5 and HA-Atg12 for 24h after saponin extraction with anti-HA antibody (red). DAPI stained nuclei are in blue. f. Immunostaining of HeLa cells transfected with control (with or without 3-MA) or DN-Rab5 for 24h after saponin extraction with anti-Atg12 antibody (red). DAPI stained nuclei are in blue. Punctate Atg12 structures in control cells are shown in arrow. Quantitation of the percentage of cells with the punctate Atg12 structures in control, 3MA or DN-Rab5 is shown in graph. *** - P < 0.0001.

Aberrant conjugation of Atg5 with Atg12 after Rab5 inhibition. a. HeLa cells transfected with control or Atg7 siRNA for 48h were subjected to second round of transfection with GFP-Atg5 together with the siRNA for another 24h. Then the cells were fixed following saponin extraction to visualise the GFP-Atg5 structures (green). b. HeLa cells transfected with control vector or DN-Rab5 together with HA-Atg12 and Atg5 were subjected to western blot analysis with anti-HA antibody to detect free Atg12 and the Atg5-Atg12 complex. The experiment assays the proportion of transfected Atg12 that gets conjugated to Atg5 by assessing the ratio of Atg5-Atg12 complex to free Atg12 as shown in the graph. Atg5-Atg12/Atg12 ratio from 4 independent experiments is shown *** - P < 0.0001. c. Similar experiments to Fig. 3b were performed in the presence or absence (control) of 3-methyladenine (3-MA; left) or with control or Vps34 siRNA (right). Atg5-Atg12/Atg12 ratio from 3 (for Vps34 siRNA) and 4 (for 3MA) independent experiments is shown. *** - P < 0.0001, ** - P < 0.001. d. HeLa cells transfected simultaneously with Rab5a, Rab5b and Rab5c siRNA (Rab5; left), Atg7 (right) or control siRNA for 48h were subjected to second round of transfection with Atg5 and HA-Atg12 together with the siRNA for another 24h. Western blot analysis was performed with anti-HA antibody to detect free Atg12 and the Atg5-Atg12 complex. Representative images from two independent, reproducible experiments are shown. e. Immunostaining of HeLa cells transfected with control (with or without 3-MA) or DN-Rab5 and HA-Atg12 for 24h after saponin extraction with anti-HA antibody (red). DAPI stained nuclei are in blue. f. Immunostaining of HeLa cells transfected with control (with or without 3-MA) or DN-Rab5 for 24h after saponin extraction with anti-Atg12 antibody (red). DAPI stained nuclei are in blue. Punctate Atg12 structures in control cells are shown in arrow. Quantitation of the percentage of cells with the punctate Atg12 structures in control, 3MA or DN-Rab5 is shown in graph. *** - P < 0.0001.