Lipid Droplets, Perilipins and Cytokeratins – Unravelled Liaisons in Epithelium-Derived Cells
Figure 5
Summarized results of immunoprecipitations (IPs) using OA treated PLC cells and PLIN antibodies followed by MS analysis.
Proteins from gradient separations as seen in scheme of Fig. 3 and in Fig. 4C of the top layer LD fraction (LD1; 0–5% iodixanol), combined fractions LD2 (5–15% iodixanol) and combined fractions LD3 (15–25% iodixanol) were used for precipitation. IPs obtained with mabs TIP47, MLDP-382, AP125 and as a control VE-cadherin were analysed by SDS-PAGE and silver staining respectively. Positions of molecular weight marker proteins are given on the left margin. For MS analysis visible bands from fractions, differing from bands seen in unbound lysate supernatant material as well as from control antibody, were chosen (for annotation of individual gel bands used for MS analysis and for detailed lists of identified proteins see Fig. S4. A survey of major MS results obtained is given in the lower part of the figure. Note, besides members of the PLIN family, especially cytokeratins 8 and 18 could be precipitated. In addition, specific enzymes and proteins involved in lipid metabolism and endocytosis could be detected - predominantly seen with TIP47 antibodies and in fractions of higher densities, including sterol reductases, fatty acyl-CoA reductase, acyl-CoA/cholesterol transferase, serine palmitoyl transferase and N-acylneuraminate cytidylyltransferase.