Fig. 3. Methylation enhances the distribution of FMRP in dense granules. (A) Western blot of HeLa cell proteins that pellet through 50% sucrose (lanes 1, 2) or pellets from HeLa cells that were treated for 24 hours with 20 µM AdOx (lanes 4, 5). Proteins in lanes 2 and 4 were from pellets treated with 25 mM EDTA prior to sedimentation. Lanes 3 and 6 are total HeLa cell extracts (20% v/v of the pellet). Blots were probed with anti-FMRP mAb, anti-PABP pAb, anti-Hsp70cp mAb, and anti-EF-1A and anti-PRMT1 mAbs. Boxed lanes are for presentation purposes. The lower panel shows Coomassie staining of the proteins in each fraction. (B) Densitometric analysis of the ratio of FMRP in the pellet fraction of EDTA-treated lysates (E) to FMRP in the pellet fraction of non-treated lysates (P) in the presence or absence of 20 µM AdOx. The results of three sets of cell lysates are presented. (C) Western blot analysis of cell lysates prepared as in A, but treated or not treated with RNAse A prior to sedimentation. Lanes 1 and 4 represent total cell proteins (20% v/v of the pellet), lanes 2 and 5 are pellet proteins treated with RNAse A, lanes 3 and 6 are untreated pellet proteins. Boxed lanes are for presentation purposes. (D) Densitometric analysis of three sets of cell lysates prepared in C.