1. Biochemical Characterization of Arylsulfatase E and Functional Analysis of Mutations Found in Patients with X-Linked Chondrodysplasia Punctata 
Aurora Daniele, Giancarlo Parenti, Marilena d'Addio, Generoso Andria, Andrea Ballabio, Germana Meroni 
The American Journal of Human Genetics 
Volume 62, Issue 3, Pages (March 1998)
DOI: /301746
Copyright © 1998 The American Society of Human Genetics Terms and Conditions

2. Figure 1
a, Immunoprecipitation and SDS-PAGE analysis of COS cells transfected with pcDL-ARSE. Mock (left) and transfected (right) Cos7 cells were labeled with 35S-methionine for 30 min; ARSE-related polypeptides were immunoprecipitated, and the immunocomplexes were analyzed by SDS-PAGE. Two bands, of 60 and 68 kD, were detectable in the Cos7 cells transfected with the ARSE cDNA. b, Biosynthesis and stability of the ARSE gene product. Cos7 cells transfected with pcDL-ARSE were labeled for 5 min and were chased for variable periods. Cell extracts were immunoprecipitated, and the immunocomplexes were analyzed by SDS-PAGE. After 5 min of labeling, only the 60-kD polypeptide was detectable. This polypeptide was rapidly converted into the 68-kD form, which is the only molecular form visible after 1, 4, and 8 h of chase; after 20 h, the 68-kD polypeptide was barely detectable. c, Glycosylation of the ARSE gene product. Cos7 cells transfected with pcDL-ARSE were labeled for 30 min and were chased for 4 h. Cell extracts were immunoprecipitated; the immunocomplexes were denatured and subjected to endoglycosidase treatment and were analyzed by SDS-PAGE, together with the untreated immunocomplexes. After endoglycosidase H treatment, the 68-kD polypeptide was converted into the 60-kD form.
The American Journal of Human Genetics  , DOI: ( /301746)
Copyright © 1998 The American Society of Human Genetics Terms and Conditions

3. Figure 2
Localization of ARSE in transfected cells. Cos7 cells were transfected with ARSE (a–d) and ARSD (e and f). NIH3T3 and RPE cells were transfected with ARSE (g and h). a and b, Double staining using anti-ARSE antiserum, followed by FITC anti-rabbit antibody, and rhodamine-conjugating WGA, respectively (× 950). c and d, Double staining using anti-ARSE antiserum, followed by TRITC anti-rabbit antibody, and monoclonal anti-Golgi 58K protein, followed by FITC anti-mouse antibody, respectively (× 380). e and f, Double staining using anti-ARSD antiserum, followed by TRITC anti-rabbit antibody, and monoclonal anti-Golgi 58K protein, followed by FITC anti-mouse antibody, respectively (× 380). g and h, Single staining using anti-ARSE antiserum, followed by TRITC anti-rabbit antibody (× 950).
The American Journal of Human Genetics  , DOI: ( /301746)
Copyright © 1998 The American Society of Human Genetics Terms and Conditions

4. Figure 3
Immunoblot of ARSE-transfected Cos7 cells solubilized with either Na2CO3 (lanes 1 and 2) or Triton X-114 (lanes 3 and 4). Lane 1, Supernatant after extraction with Na2CO3. Lane 2, Pellet. After extraction with Triton X-114, the extract was separated into the aqueous (lane 3) and detergent (lane 4) phases.
The American Journal of Human Genetics  , DOI: ( /301746)
Copyright © 1998 The American Society of Human Genetics Terms and Conditions

5. Figure 4
A, Immunoblot analysis of ARSE mutants. Total extracts of cells transfected with the ARSE wild-type and mutant cDNAs were subjected to SDS-PAGE, followed by immunoblotting with anti-ARSE antiserum. B, Biosynthesis and stability of the wild-type and G137V mutant ARSE. Cos7 cells transfected with the constructs carrying either the wild-type or the mutated ARSE cDNA were labeled for 30 min and were chased for variable periods. Cell extracts were immunoprecipitated, and the immunocomplexes were subjected to SDS-PAGE analysis; the results obtained with the construct carrying the mutant ARSE cDNA are shown (right) as compared with the results obtained with the wild-type cDNA (left). C, R12S ARSE-transfected Cos7 cells stained with anti-ARSE antiserum, followed by TRITC anti-rabbit antibody.
The American Journal of Human Genetics  , DOI: ( /301746)
Copyright © 1998 The American Society of Human Genetics Terms and Conditions