Blockade of 4-1BB and 4-1BBL Interaction Reduces Obesity-Induced Skeletal Muscle Inflammation
Figure 3
Expression of inflammatory cytokines in primary myotubes treated with 3E1. Three-day primary myotubes derived from 4-week-old WT and 4-1BB-deficient mice. (a) Representative bands of 4-1BB mRNA were determined by semiquantitative RT-PCR. Primary myotubes were pretreated with 10 ng/mL TNF in DMEM containing 0.1% FBS for 12 h. Then the cells were washed twice with PBS and given 10 g/mL 3E1 or 10 g/mL rat IgG (control) in serum-free DMEM for 24 h (b–f) or 3 h (g). (b–d) qRT-PCR analysis of mRNA for the inflammatory cytokines TNF, IL-6, and MCP-1. ((e) and (f)) Levels of release of inflammatory cytokines IL-6 and MCP-1 in the culture media were measured by ELISA. (g) Expression of phosphorylated IKK/β, IKKβ, IκB, and -tubulin proteins was determined by Western blotting using the indicated antibodies (lower panel). Band intensities were measured densitometrically using ImageJ. Relative intensities of the bands were displayed as fold of control (upper panels). Data are means ± SEM of three independent triplicate experiments. *, **, and *** significantly different between 3E1- and rat IgG-treated groups or between WT and KO groups.
