Fig. 7. Cell fractionation shows that AAM-B is targeted to droplets. (A) Protease-protection assay shows that AAM-B is exposed to the cytosol. HeLa cells were transfected with GFP-AAM-B-Myc and equal aliquots of the total membranes were incubated in the absence (lane 1) or presence (lanes 2 and 3) of trypsin. Lane 3 contained a protease inhibitor. An immunoblot for calnexin (Cnx) shows that the protein is reduced by 10 kDa, which corresponds to the size of the cytosolic part of the protein. GFP and Myc are digested in the presence of trypsin, demonstrating that they are cytosolically oriented. (B-F) COS7 cells were transfected with cDNAs encoding Myc-tagged AAM-B constructs and cell fractions prepared. Equal volumes of the droplet, cytosol and membrane fractions were separated by SDS-PAGE. (B) A representative gel was stained with Coomassie Blue to determine the relative protein load for the immunoblots in C-F. (C-F) Each preparation was immunoblotted with -Myc, -ADRP, -Bip, -Sec61β, -PDI and -actin IgG. (C) The wild-type protein was found in both droplet and membrane fractions. (D) The N mutant was found solely in the membrane fraction. (E) The C6 mutant was found in the droplet fraction. (F) The Jxm mutant was found in the membrane fraction.