Supplemental Figure S1
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Fig. S1. The loss of elongated GSLs does not impair Grk secretion but modifies its recognition by the anti-Grk antibody. Confocal images of stage 10a egg chambers labeled by immunofluorescence for Grk (red). Anterior is to the left and dorsal is up. Prior to fixation and processing for conventional staining, chambers were preincubated in Schneider’s medium supplemented with 10% fetal bovine serum either at 4°C for 1 hour and 30 minutes (A-D′) or at 25°C for 1 hour (E-H′). Alternatively, chambers were directly processed for conventional staining, but detergent was omitted (I-L′); in this case, only extracellular Grk is visualized since the absence of detergent prevents antibody penetration into the tissue. This technique also yields higher background in follicle cells. In wild-type egg chambers (A-B′,E-F′,I-J′), none of these treatments significantly altered Grk distribution (shown alone in enlargements of the DA region of the oocyte and follicular epithelium in B′,F′,J′; compare with conventional staining in Fig. 4G′). In brn mutants (C-D′,G-H′,K-L′), whereas Grk could not be seen in the extracellular space by conventional staining (Fig. 4I′), it became detectable when detergent was omitted or when the chambers were preincubated in serum at 4°C, but not at 25°C (see enlargements of the DA region for Grk alone in L′,D′,H′, respectively). These results indicate that Grk is secreted in the brn mutant context, but that its detection has become temperature- and detergent-sensitive.
