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Residues SFQ (173-175) in the large extracellular loop of CD9 are required for gamete fusion

Guo-Zhang Zhu1,*, Brent J. Miller2, Claude Boucheix3, Eric Rubinstein3, Christopher C. Liu4, Richard O. Hynes4, Diana G. Myles2 and Paul Primakoff1

1 Department of Cell Biology and Human Anatomy, School of Medicine, University of California Davis, Davis, CA 95616, USA
2 Department of Molecular and Cellular Biology, University of California Davis, Davis, CA 95616, USA
3 Institut National de la Sante et de la Récherche Medicale (INSERM), Unite 268, Hôpital Paul-Brousse, 94800 Villejuif, France
4 Howard Hughes Medical Institute, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA



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  		Fig. 1. Western blot of GST-EC2 and EC2/HT. Western blotting was carried out as described in Materials and Methods. Lane 1, 50 ng GST-EC2; lane 2, 10 ng EC2/HT. The molecular mass (kDa) of protein standards is indicated.

 


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  		Fig. 2. Immunofluorescent staining of CD9 on the surface of CD9–/– eggs injected with either mouse or human CD9 mRNAs. (A) Immunofluorescent staining using the anti-mouse CD9 antibody KMC8 on wild-type eggs. (B) Immunofluorescent staining using the anti-mouse CD9 antibody KMC8 on CD9–/– oocytes injected with mouse CD9 mRNA. (C) Immunofluorescent staining using the anti-human CD9 antibody ALB6 on CD9–/– oocytes injected with human CD9 mRNA.

 


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  		Fig. 3. CD9-eGFP fluorescence observed for CD9–/– oocytes injected with either wild-type or F174A mutant mouse CD9-eGFP mRNA. (A) Fluorescence observed in CD9–/– eggs injected with wild-type CD9-eGFP mRNA. (B) Fluorescence observed in CD9–/– eggs injected with F174A mutant CD9-eGFP mRNA. In A and B, both intracellular and surface fluorescence are observed. (C) Fluorescence observed in uninjected CD9–/– eggs. (D-F) Phase contrast images of A-C, respectively. Fluorescence levels were somewhat variable within each group of eggs injected with the identical amount and type of mRNA.

 


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  		Fig. 4. Western blot assay of CD9-eGFP expression in CD9–/– eggs injected with either wild-type or F174A mutant mouse CD9-eGFP mRNA. The injected eggs were allowed to develop to the M-II phase, then were lysed in non-reducing SDS-sample buffer. Western blots were carried out as described in Materials and Methods. Each lane contains 20 eggs. Lane 1, CD9–/– eggs; lane 2, CD9–/– eggs injected with wild-type CD9-eGFP mRNA; lane 3, CD9–/– eggs injected with mutant CD9- F174A-eGFP mRNA. The molecular mass (kDa) of protein standards is indicated.

 


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  		Fig. 5. Immunofluorescent staining of CD9 on the surface of CD9–/– eggs injected with either wild-type or SFQ(173-175)AAA mutant mouse CD9 mRNA. After in vitro fertilization, eggs were fixed and washed as described in Materials and Methods. The fixed eggs were incubated with anti-mouse CD9 antibody KMC8 as the primary antibody, followed by a goat anti-rat IgG (H+L) conjugated with Alexa FluorTM 488 as the secondary antibody. Immunofluorescent staining was obtained with a laser scanning confocal microscope (Carl Zeiss, LSM 410). (A) Immunofluorescent staining on CD9–/– eggs injected with wild-type mouse CD9 mRNA. (B) Immunofluorescent staining on CD9–/– eggs injected with SFQ-to-AAA mutated mouse CD9 mRNA. (C,D) Phase contrast images of A and B, respectively. Fluorescence levels were somewhat variable within each group of eggs injected with the identical amount and type of mRNA.

 


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  		Fig. 6. Western blot assay of CD9 expression in CD9–/– eggs injected with either wild-type or mutant CD9-SFQ(173-175)AAA mRNA. The injected eggs were allowed to develop to the M-II phase, then were lysed in non-reducing SDS-sample buffer. Western blots were carried out as described in Materials and Methods. Each lane contains 20 eggs. Lane 1, CD9–/– eggs; lane 2, CD9–/– eggs injected with wild-type CD9 mRNA; lane 3, CD9–/– eggs injected with mutant CD9-SFQ(173-175)AAA mRNA. The molecular mass (kDa) of protein standards is indicated.

 

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© The Company of Biologists Ltd 2002