Figure 2.

Cytokine mixture treatment increases recruitment of cytokine receptors into caveolae in INS-1 cells. INS-1 cells were incubated for 24 h with the cytokine mixture (CM; IL-1β 20 ng/ml, TNFα 20 ng/ml) and sucrose density gradient fractions were obtained from the cell lysates. Fractions are numbered 1 through 12 from top to bottom. (a) Protein levels in each fraction from CON and CM treated cells. (b) Westernblot with anti-cav-1, anti-IL-1β receptor (IL-1R1), anti-TNF receptor (TNFR), and anti-flotillin-1 antibodies. Flotillin-1 was used as a lipid raft marker. (c) The colocalization of IL-1R1, TNFR and cav-1 was confirmed by confocal microscopy. Nuclei were counterstained with DAPI (Blue). (d) Colocalization intensity was measured by Image J. Scale bars, 200 μm. Values are means ± SEM from triplicate experiments. *p < 0.05 vs. CON.