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. 2012 Nov 7;448(Pt 2):189–200. doi: 10.1042/BJ20120339

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© 2012 The Author(s) The author(s) has paid for this article to be freely available under the terms of the Creative Commons Attribution Non-Commercial Licence (http://creativecommons.org/licenses/by-nc/2.5/) which permits unrestricted non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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(a) STIM1. Left-hand panels: Western blot for STIM1 (ProSci 4119); lane 1: human sperm proteins purified by immunoprecipitation with an anti-STIM1 antibody. A band is seen at ~95 kDa and also at 55–60 kDa due to the presence of anti-STIM1 antibody from the immunoprecipitation procedure. Lane 2 is protein from STIM1–GFP-transfected HEK-293 cells. STIM1 appears at ~110 kDa due to the presence of the 25 kDa GFP tag. Separation of images in this and other gels indicates that lanes were not originally directly adjacent. Right-hand panels: immunofluorescent staining with anti-STIM1 antibody (ProSci). Upper panels show STIM1 staining and the corresponding phase image. Fluorescence occurs over the midpiece with a bright spot at the sperm neck (arrows). Lower panels show cells incubated with antibody pre-adsorbed with the antigenic peptide, which abolished staining. (b) STIM2. Left-hand panels: Western blot for STIM2 (ProSci antibody 4123); lane 1: human sperm proteins. An intense doublet is present at 85–90 kDa. Lane 2: as lane 1, but antibody was pre-adsorbed with the antigenic peptide. Right-hand panels: immunofluorescent staining with anti-STIM2 antibody. The upper panels show STIM2 staining and corresponding phase image. Staining occurs over the flagellum, being heaviest at the midpiece (white arrows). In a minority of cells (<10%), we observed staining over the acrosome (yellow arrow). The lower panels show cells incubated with antibody pre-adsorbed with the antigenic peptide, which abolished flagellar and acrosomal staining but resulted in fluorescence just behind the equatorial segment (blue arrows). (c) Orai 1. Left-hand panels: Orai 1 immunoblot (Sigma antibody O8264); lane 1: human sperm proteins. Lane 2: proteins extracted from Myc-tagged Orai 1-transfected HEK-293 cells. Deduced molecular mass of non-glycosylated Orai 1 is ~35 kDa. Right-hand panel: immunofluorescent staining with anti-Orai 1 antibody. Upper panels show Orai 1 staining (Sigma antibody O8264) and corresponding phase image. Staining occurs primarily over the acrosome and midpiece and weakly on the principal piece. Lower panels show cells stained similarly but omitting the primary antibody. (d) Orai 2. Left-hand panels: Western blot for Orai 2 (ProSci antibody 4111); lane 1: human sperm proteins. Lane 2: as lane 1, but antibody pre-adsorbed with the antigenic peptide. Right-hand panel: immunofluorescent staining with anti-Orai 2 antibody. Upper panels show Orai 2 staining and corresponding phase image. Staining occurs over the midpiece (white arrows) and acrosome (yellow arrows), with weaker staining over the principal piece. Lower panels show cells incubated with antibody pre-adsorbed with the antigenic peptide, which reduces/abolishes staining of the acrosome, midpiece and flagellum, but resulted in fluorescence just behind the equatorial segment (blue arrows). (e) Orai 3. Left-hand panels: Western blot for Orai 3 (ProSci antibody 4215). Lane 1: human sperm proteins. Lane 2: as lane 1, but antibody was pre-adsorbed with the antigenic peptide, which did not block band detection. Right-hand panels: immunofluorescent staining with anti-Orai 3 antibody. Upper panels show Orai 3-staining and corresponding phase image. Staining occurs primarily over the anterior midpiece and sperm neck (arrows). Lower panels show cells incubated with antibody pre-adsorbed with the antigenic peptide, which abolished staining. (f) Diagrammatic representation of ‘typical’ localization (immunofluorescence pattern) for each of the proteins investigated.