Fig. 5.

Further validation of TRIO, CHKA and BMX involvement in AT₁R–EGFR transactivation. (A,B) HMEC-LST-AT₁R cells were reverse transfected with 40 nM Dharmacon siGENOME SMARTpool siRNAs targeting TRIO, CHKA or BMX expression. HMEC-LST-AT₁R cells transfected with TRIO siRNA (siTRIO) demonstrated an approximate 45% knockdown of mRNA transcript at 24 hours, by qRT-PCR (A) and at 72 hours post-transfection, knockdown of TRIO reduced AT₁R–EGFR transactivation upon stimulation with AngII (B). (C,D) When cells were transfected with CHKA siRNA (siCHKA), an approximate 90% knockdown of CHKA transcript was observed at 24 hours (C), coupled with a reduction in ChoK (CHKA) protein expression and AT₁R–EGFR transactivation at 72 hours (D). (E,F) Similarly, cells transfected with BMX siRNA (siBMX) demonstrated an approximate 90% knockdown of mRNA transcript at 24 hours (E), and a reduction in AT₁R–EGFR transactivation (as determined by phospho-EGFR and phospho-ERK1/2 abundance) was observed after 72-hour siRNA knockdown (F). n = 3 or 4 experiments for both mRNA and protein analysis; for mRNA analysis *P = 0.0144 (TRIO), ***P = 0.0002 (CHKA) and P = 0.0008 (BMX), paired two-tailed Student's t-test. Western blots are representative of all experiments.