Fig. 6

BMS777607 (BMS) treatment prevented ubiquitination of AXL. a Co-immunoprecipitation experiments of AXL and ubiquitin by using anti-ubiquitin antibody FK2 in Hs578T cell lysates after 1 h of BMS treatment and 250 ng/ml exogenous GAS6 ligand stimulation for 15 min are shown. The amount of precipitated AXL was subsequently determined by western blot. Ubiquitinated AXL was detectable as smear at high molecular weight in DMSO treated control lysates. (b) GAS6 stimulation increased the amount of ubiquitinated AXL significantly, in contrast to BMS, which blocked the ubiquitination completely. AXL western blot after treatment with 0.5 μM BMS of (c) Hs578T and (d) H292 cells which were transiently transfected with HA-tagged AXL gatekeeper mutant (K567R) and wildtype control. Exogenous AXL was quantified by anti-HA antibody and total AXL by a C-terminally binding anti-AXL antibody (H-3). 120 kDa AXL was used for normalization to consider transfection efficacy. BMS increased AXL 140kD/120kD protein ratio in wildtype overexpressing (c) Hs578T to fivefold and to twofold in (d) H292 cells. AXL gatekeeper mutant (K567R) is not responsive to BMS treatment, although exhibiting a similar AXL protein ratio compared to the wildtype transfected and BMS treated cells. (e) Representative western blots are displayed for Hs578T and H292 cells. Mean values and SEM of three independent experiments are shown. Differences with *P < .05, **P < .01, and ***P < .001 were considered statistically significant