Figure 2. PPXY motifs are required for Shh-induced endocytosis of Ptch1.
(A) Confocal images showing colocalization of Ptch1-GFP or Δ2PY (green) with Rab7-RFP (red), and (B) calculation of the colocalization coefficients in (A) in transfected MEFs. (C) Confocal images showing localization of Ptch1-GFP or Δ2PY (green) in vesicles marked anti-Lamp1 (red) in the presence of 1 mg/ml leupeptin. (D) Confocal imaging and (E) calculation of colocalization coefficient of Ptch1-GFP and Rab7-RFP in Kif3a−/− and control MEFs. ShhN treatment was for 1 hr and the cells were processed as in Figure 1A. Statistical analyses were performed by two-tail Student's t test. ***p<0.001, and n.s., not statistically significant (p>0.05).
Figure 2—figure supplement 1. Shh promotes colocalizaiton of Ptch1-GFP with endogenous Rab7 in late endosomes.
Representative confocal images showing ShhN treatment promotes
colocalization of Ptch1-GFP in late endosomes visualized by
anti-Rab7. Transfected MEFs were treated with ShhN-CM or control
conditioned medium for 1 hr, followed by incubations at 4°C
for 20 min and 37°C for 20 min. The close-up images were
digitally amplified.
Figure 2—figure supplement 2. Lack of colocalization of Ptch1-GFP or Δ2PY-GFP with exogenous Rab5-RFP and Lamp1-RFP without blocking lysosomal turnover.
Representative Confocal images and quantification of
colocalization coefficients showing that Ptch1-GFP or
Δ2PY-GFP does not colocalize with Lamp1-RFP (red)
(A) or Rab5-RFP (B). Transfected
MEFs were treated ShhN or control conditioned medium without
Leupeptin for 2 hr, and then the cells were processed as in
Figure 2A.
Figure 2—figure supplement 3. Ptch1-GFP or Δ2PY-GFP was not found in early endosomes marked by anti-Rab5 immunofluorescence staining.
Representative Confocal images showing Ptch1-GFP or
Δ2PY-GFP and endogenous Rab5 in non-overlapping green or
red channel, respectively.