Fig. 4. Symmetrical methylation of SmB and SmD3 proteins is dependent on Csul and Vls. (A) Csul and Vls are required for sDMA synthesis. Protein extracts of wild-type (WT), csul^RM, vls¹, osk⁸⁴/pXT103, vas^Q7, vas^O11 and aub^HN2/aub^N11 ovaries were separated by SDS-PAGE, blotted and probed with -SYM10 (left panel) and -ASYM24 antibodies (right panel). Anti-ribosomal p40 antibodies (-p40) were used as a loading control (left panel, lower blot). The intensity of four SYM10-reactive protein bands in the range of 15 to 26 kDa was strongly reduced in csul^RM and vls¹ protein extracts. No change of the intensity of the ASYM24-reactive bands was observed in any mutant background. [B] and [C] indicate the position of protein bands shown in B and C, respectively. (B) SmB is symmetrically dimethylated in vivo. Ovarian protein extracts of wild-type and SmB^BG02775/CyO females were separated by SDS-PAGE, blotted and probed with -SYM10 and -Y12 antibodies. By comparison to wild type, the intensity of the SYM10/Y12-reactive protein band is significantly reduced in SmB^BG02775/CyO. (C) SmD3 is symmetrically dimethylated in vivo. Protein extracts of wild-type and homozygous SmD3^l(2)k131-07 larvae were separated by SDS-PAGE, blotted and probed with -SYM10 antibodies. Arrow indicates the position of SmD3. (D) SmB immunostaining using -Y12 antibodies. sDMA-SmB localizes in the nuclei of the nurse cells and somatic follicular cells of wild-type egg chambers but is undetected in csul^RM egg chambers.