PLIN proteins and vimentin detected in different density gradient centrifugation fractions of briefly AIM-stimulated preadipocytes.
(a) Fractions obtained by cell disintegration using nitrogen cavitation, iodixanol gradient centrifugation followed by SDS-PAGE separations and Coomassie blue staining are shown (cp. ). Separated fractions were tested with Western blotting (WB) using pabs TIP47-hCT(b), S3-12-hNT (c,c), Adipo-hCT (d,d) and mabs Peri112.17 (e,é) and Vim3B4 (f,f´). Positions of molecular weight markers are given on the left margin of (a) and fraction numbers on top of (a,b). (c,d,é,f´) represent prolonged exposures of reactions shown in (c,d,e,f) respectively. The higher molecular band reactions of adipophilin and perilipin seen in LD1 fractions are unknown modifications of PLIN proteins (asterisks in d,e). Note, the separation shown here is given with AIM-stimulated preadipocytes and not with AIM-stimulated plus OA-treated preadipocytes. Therefore only the few small (i.e. freshly endocytosed) LDs are detected. These are mostly coalesced with the bigger endogenously generated LDs and therefore preferentially found here within the LD1 fraction. Importantly PLIN proteins can be detected also in these non OA-treated preadipocytes together with vimentin within all three major LD gradient areas LD1, LD2, LD3 - including the top layer LD1.