Fig. 6

Development of an antibody targeting endogenous MASC protein and RIP-qPCR analysis. The specificity of the developed anti-MASC antibody was evaluated by western blotting and immunohistochemistry. a Western blottings using the anti-MASC antibody and an anti-DSX-DBD antibody used in the previous study [26] were performed with whole protein extracts from gonads of day-3 5th instar larvae. Sizes of the molecular markers are indicated on the left. Histone H3 protein levels were used as loading control (Additional file 9, right panel, lanes 1 and 2). b Cross-section illustration of the testis from a 5th instar larva. The testicular basement membrane is shown in gray. The tissue area shown in the immunostained image is indicated by a red box. c Frozen testis sections from day-3 5th instar larva were stained with a Masc-specific antisense riboprobe or with its sense strand probe (upper panel). The same ISH was performed using a Bmdsx-specific antisense riboprobeor its sense strand probe (lower panel). d RNA/MASC protein complexes were immunoprecipitated using the anti-MASC antibody, followed by qRT-PCR. The relative enrichment was defined as the level of qPCR product amplified from samples relative to that from samples precipitated using a negative control antibody (anti-rabbit IgG antibody). Values are presented as the mean ± SE from three independent qPCR assays. Results are representative of three independent experiments. * indicates a significant difference (< 0.05) compared with the negative control, as determined by Welch’s t-test. e Immunostaining using anti-MASC antibody was used to examine whether the antibody detected localization of the endogenous MASC protein in the testis. f The same section of the testis was subjected to immunostaining using an anti-DSX-DBD antibody. g Merged laser confocal microscopy image showing MASC (red) and BmDSX (blue) expression. h Nuclei were counterstained with DAPI. The dotted line indicates the testicular basement membrane. i–l High magnification images of testis sections immunostained with anti-MASC (i) or anti-DSX-DBD antibody (j). k Merged image showing MASC (red) and BmDSX (blue) expression. Arrowheads indicate MASC and BmDSX co-localization. (l) Nuclei were counterstained with DAPI