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. Author manuscript; available in PMC: 2013 Sep 11.
Published in final edited form as: Sci Signal. 2013 May 7;6(274):ra32. doi: 10.1126/scisignal.2003741

Fig. 4. CAS degradation in lapatinib-resistant cells.

Fig. 4

(A) BT474 and rBT474 (left) cultured in the absence of lapatinibfor1weekwere treated with lapatinib for the indicated times and immunoblotted for CAS and actin (top). Densitometric analysis of the abundance of CAS normalized to that of actin from four independent experiments (means ± SEM) is shown (bottom). CAS abundance at time 0 in BT474 cells was set at 100%. (B) rBT474 cells maintained in the presence of lapatinib were harvested at various time points after removal of lapatinib and immunoblotted for CAS and actin (top). Densitometric analysis of the abundance of CAS normalized to that of actin from four independent experiments (means ± SEM) is shown (bottom). CAS abundance at time 0 was set at 100%. (C) BT474 and rBT474 cells expressing FLAG-tagged CAS were treatedwith cycloheximide(CHX)andlapatinibfortheindicated times and immunoblotted for FLAG and actin (top). Densitometric analysis of the abundance of FLAG-CAS normalized to that of actin from four independent experiments (means ± SEM) is shown (bottom). FLAG-CAS abundance at time 0 in each cell line was set at 100%. (D) BT474 and rBT474 expressing FLAG-CAS and HA-Ub were treated with lapatinib and z-VAD and harvested after MG132 treatment. FLAG-CAS immunoprecipitates were immunoblotted for HA to analyze ubiquitylation of CAS. n = 2 independent experiments. (E) H1299 cells were transfected with empty vector, FLAG-CAS WT, or FLAG-CASW127A. Immunoprecipitates of the FLAG-tagged proteins were immunoblotted for MDM2. Densitometric analysis of the abundance of immunoprecipitated MDM2 normalized to that of FLAG-CAS WT or FLAG-CASW127A from four independent experiments is shown (means ± SEM). The amount of MDM2 bound to FLAG-CAS WT was set at 100% (*P < 0.05 by paired, two-tailed t test). (F) BT474 and rBT474 cells expressing FLAG-CAS were treated with lapatinib and z-VAD and harvested after MG132 treatment. FLAG-CAS immunoprecipitates were immunoblotted to detect association of endogenous MDM2. Densitometric analysis of the abundance of immunoprecipitated MDM2 normalized to that of immunoprecipitated FLAG-CAS is shown (means ± SEM; n = 4 independent experiments). The immunoprecipitated MDM2 in BT474 cells without any treatments was set at 100% (*P < 0.05 by paired, two-tailed t test). (G) H1299 cells expressing empty vector, FLAG-CASW127A, or FLAG-CAS WT were treated with MG132. Ubiquitylation of CAS was analyzed after immunoprecipitation. n = 2 independent experiments. (H) H1299 cells expressing MDM2 (WT, C/A mutant, or empty vector), FLAG-CAS (WT or F123A mutant), and GFP (transfection control) were immunoblotted for FLAG, MDM2, GFP, and actin. n = 3 independent experiments.