Fig 1.
Colocalization between LAMP1 and Rab11 based on different means of ectopic expression.
(A) Representative confocal image of HeLa wild-type cells transfected with both DsRed-Rab11 and LAMP1-GFP. (B) Representative confocal image of wild-type HeLa cells transiently transfected with DsRed-Rab11 with subsequent immunofluorescence staining against LAMP1. (C) Representative confocal images of HeLa cells expressing endogenously tagged GFP-Rab11 transiently transfected with LAMP1-DsRed tagged vector. (D) Representative confocal images of RFP-Rab11 KI cells transfected with LAMP1-GFP. The scalebars indicate 20 µm and the zoomed areas are indicated by squares. Arrowheads indicate the structures where colocalization occurs. (E) Quantitation of the colocalization between Rab11 and LAMP1 in the different experimental conditions shown in panels A-D (at least 40 cells per condition were scored from three independent experiments indicated by different color shades; 1403 Rab11 vesicles and 2265 LAMP1 vesicles were counted for condition A, 904 Rab11 vesicles and 908 LAMP1 vesicles were counted for condition B; 1541 Rab11 vesicles, and 965 LAMP1 vesicles were counted for condition C; 1427 Rab11 vesicles and 1653 LAMP1 vesicles were counted for condition D). The bars in the graph represent the mean and the error bars correspond to the standard deviation.
Fig 2.
Validation of the anti-LAMP1 antibody and the knock-in cells (A) Western-blot validation of the LAMP1 antibody by comparing cells treated with a control siRNA and an siRNA directed at LAMP1.
(B) Representative confocal images of LAMP1-stained HeLa wild-type cells treated with a control siRNA or an siRNA directed at LAMP1. (C) Western-blot comparison of wild-type HeLa cells vs. GFP-Rab11 knock-in cells (clone #3) and RFP-Rab11 knock-in cells (clone #9) using an anti-Rab11 antibody (middle panel) as well as an anti-GFP antibody that recognises GFP but not RFP to visualize GFP-KI Rab11 (right panel). Ponceau S Staining is shown to evaluate the evenness of loading (left panel). The asterisks show unspecific bands and the arrows point to Rab11. (D) Representative confocal images of wild-type HeLa cells, the GFP-Rab11 knock-in cells (clone #3) and the RFP-Rab11 knock-in cells (clone #9). (E) Western-blot comparison of wild-type HeLa cells vs. LAMP1-mKusabiraOrange2 and GFP-Rab11 double knock-in cells using an anti-LAMP1 antibody. Whole protein loading is shown (Ponceau S Staining) as well as an anti-β-actin staining. (F) Representative confocal images of LAMP1-stained LAMP1-mKusabiraOrange2 knock-in cells treated with a control siRNA or an siRNA directed at LAMP1. Scalebars: 20 µm.
Fig 3.
Absence of colocalization between LAMP1 and Rab11 when studied at their endogenous levels.
(A) Representative confocal image of immunofluorescence staining using an anti-LAMP1 antibody (visualized with an AlexaFluor647-conjugated secondary antibody) in GFP-Rab11 cells. (B) Representative confocal image of LAMP1-mKusabiraOrange2 / GFP-Rab11 knock-in cells. Scalebars: 20 µm. Enlarged areas are indicated by squares. Arrowheads indicate the structures where colocalization occurs. (C) Quantitation of the colocalization between Rab11 and LAMP1 in the different experimental conditions shown in panels A-B (at least 45 cells per condition were scored from three independent experiments; 1809 Rab11 vesicles and 1227 LAMP1 vesicles were counted for condition A; 1266 Rab11 vesicles and 1245 LAMP1 vesicles were counted for condition B). Scale enlargement is also shown to better appreciate the very low colocalization between Rab11 and LAMP1. The bars in the graph represent the mean and the error bars correspond to the standard deviation..
Table 1.
sgRNAs used for the generation of stable Kis.