Figure 3. Sulindac Inhibits TNFα-induced AKT Activation and tRXRα-p85α Interaction.

(A) Inhibition of AKT activation by Sulindac. The indicated cells starved overnight and treated with Sulindac (100 μM) for 1 hr were analyzed for AKT activation by immunoblotting.

(B) Inhibition of basal AKT activation by RXRα siRNA. HepG2 cells transfected with RXRα siRNA for 48 hr were treated with Sulindac (100 μM) for 1 hr. AKT activation and RXRα expression were analyzed by immunoblotting.

(C) Inhibition of TNFα-induced AKT activation by Sulindac and RXRα siRNA. A549 lung cancer cells transfected with RXRα or control siRNA for 48 hr were pretreated with Sulindac (100 μM) for 1 hr before exposed to TNFα (10 ng/ml) for 30 min.

(D) Synergistic inhibition of AKT activation by TNFα and Sulindac. ZR-75-1 and PC3 cells were pretreated with Sulindac for 1 hr before exposed to TNFα (10 ng/ml) for 30 min.

(E) Induction of RXRα-p85α interaction by TNFα. A549 cells treated with TNFα (10 ng/ml) and/or Sulindac (100 μM) for 30 min were analyzed for RXRα-p85α interaction by co-immunoprecipitation using ΔN197 antibody. Above, schematic representation of anti-RXRα antibodies used in co-immunoprecipitation and immunoblotting assays. D20 antibody recognizes amino acids 2–21 in the N-terminal A/B domain, while ΔN197 antibody recognizes the C-terminal E/F domain. A RXRα truncated protein with about 44 kDa is also shown.

(F) Expression of tRXRα in various cancer cell lines. The indicated cell lines treated with or without 9-cis-RA (10⁻⁷ M) for 30 min were analyzed by immunoblotting using the ΔN197 RXRα antibody.

One of three to six similar experiments is shown.

See also Figure S3.