Figure 2. Down-regulated Turnover of AXL in Gefitinib Resistant H292 (H292-Gef) Cell Line.

(A) H292 and H292-Gef cells were treated with gefitinib for 72 h, and the proliferation of the cells was measured using the SRB assay. The IC₅₀ values were calculated using the TableCurve 2D software, and the data are presented as the means ± SD. (B) The basal protein expression of AXL was determined by western blot using β-actin as the loading control. (C) The cells were treated with 25 μg/ml CHX for the indicated times. The lysates were analyzed by western blot analysis with antibody against C-terminal AXL using β-actin as a loading control. The expression levels were quantified by densitometry using ImageJ. (D) The mRNA expression of the indicated markers in cells was determined by real-time PCR, and the β-actin mRNA levels were used for normalization. The data are presented as the mean fold changes ± SD relative to the H292 control. (E) H292-Gef cells were treated with GM6001 and/or compound E overnight and then with MG132 for 3 h before being collected for western blot analysis using β-actin as a loading control. For determination of NTF, the culture medium (CM) was collected, immunoprecipitated with antibody against N-terminal AXL, and immunoblotted using anti-N-terminal AXL. The results are representative of two (C, E) or three (A, B, D) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 by t-test.