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1.
FIGURE 4.

FIGURE 4. From: Nuclear Translocation of Epidermal Growth Factor Receptor by Akt-dependent Phosphorylation Enhances Breast Cancer-resistant Protein Expression in Gefitinib-resistant Cells.

Phosphorylation of EGFR at Ser-229 by Akt plays a role in the development of gefitinib resistance. The cytostatic effect of gefitinib on the A431/GR cells expressing adenoviral-derived EGFR was measured by MTT assay. Error bars denote S.E. (n = 3).

Wei-Chien Huang, et al. J Biol Chem. 2011 June 10;286(23):20558-20568.
2.
FIGURE 5.

FIGURE 5. From: Nuclear Translocation of Epidermal Growth Factor Receptor by Akt-dependent Phosphorylation Enhances Breast Cancer-resistant Protein Expression in Gefitinib-resistant Cells.

Nuclear EGFR regulates BCRP/ABCG2 expression in A431/GR cells. A, mRNA and protein expressions of BCRP/ABCG2 in A431 and A431/GR cells transfected with control or EGFR siRNA were analyzed by RT-PCR and WB, respectively. B and C, effects of Akt inhibitor VIII (B) and Akt siRNA (C) on the BCRP/ABCG2 protein expression in A431/GR cells were examined by WB. N.S., non-specific siRNA.

Wei-Chien Huang, et al. J Biol Chem. 2011 June 10;286(23):20558-20568.
3.
FIGURE 3.

FIGURE 3. From: Nuclear Translocation of Epidermal Growth Factor Receptor by Akt-dependent Phosphorylation Enhances Breast Cancer-resistant Protein Expression in Gefitinib-resistant Cells.

Phosphorylation by Akt increases EGFR nuclear translocation. A, endogenous EGFR Ser-229 phosphorylation in both cytoplasm (Non-NE) and nucleus (NE) of A431/GR cells was detected by using anti-phospho-EGFR Ser-229 for IP and subsequently anti-EGFR antibody for WB. B, Western blot analysis and quantification of nuclear EGFR expression in A431 and A431/GR cells treated with or without the Akt inhibitor, API-2, is shown. C, shown is a Western blot analysis of nuclear EGFR expression in A431 and A431/GR cells transfected with control (N.S., non-specific) or Akt siRNA. D, shown is WB analysis of nuclear EGFR expression in HEK293 cells transfected with three Akt isoforms individually. E, shown is Western blot analysis of nuclear expressions of EGFR-WT and EGFR-Ser-229 mutants in HEK293 cells co-transfected with or without HA-myr-Akt1. F, nuclear localization of EGFR-WT and EGFR-Ser-229 mutants in HeLa cells co-transfected with or without HA-myr-Akt1 was examined by confocal microscopy. Bar, 5 μm.

Wei-Chien Huang, et al. J Biol Chem. 2011 June 10;286(23):20558-20568.
4.
FIGURE 2.

FIGURE 2. From: Nuclear Translocation of Epidermal Growth Factor Receptor by Akt-dependent Phosphorylation Enhances Breast Cancer-resistant Protein Expression in Gefitinib-resistant Cells.

Akt phosphorylates EGFR at Ser-229. A, whole cell lysates prepared from cells were subjected to IP/WB analysis by using indicated antibodies. PAS, anti-phospho-Akt-substrate antibody. B, HEK293 cells were transfected with the indicated constructs and then subjected to IP/WB analysis. C, MDA-MB-468 cells were treated with EGF for indicated times, and IP/WB analysis was performed to assess the physical interaction between Akt and EGFR. D, in vivo EGFR Ser-229 phosphorylation was detected in anti-EGFR immunoprecipitates from EGF-treated MDA-MB-468 cells by mass spectrometry. E and F, substitution of Ser-229 to Ala abolished EGF- or Akt-induced EGFR phosphorylation detected by anti-PAS antibody in anti-EGFR (E) or anti-myc (F) immunoprecipitates from transfected HEK293 cells. G, endogenous EGFR Ser-229 phosphorylation was detected in A431/GR cells by using anti-phospho-EGFR Ser-229 for IP and anti-EGFR antibody for subsequent WB. H, Akt expression in A431/GR cells was deprived by Akt siRNA. Then endogenous EGFR Ser-229 phosphorylation was detected by using anti-phospho-EGFR Ser-229 for IP and anti-EGFR antibody for subsequent WB. N.C., non-specific control.

Wei-Chien Huang, et al. J Biol Chem. 2011 June 10;286(23):20558-20568.
5.
FIGURE 1.

FIGURE 1. From: Nuclear Translocation of Epidermal Growth Factor Receptor by Akt-dependent Phosphorylation Enhances Breast Cancer-resistant Protein Expression in Gefitinib-resistant Cells.

Nuclear EGFR is involved in drug resistance to EGFR-tyrosine kinase inhibitor gefitinib. A, A431 and A431/GR cells were subjected to cellular fractionation followed by WB analysis of cellular localization of EGFR. Levels of tubulin and lamin B were used as markers for cytosolic and nuclear fractions, respectively. B, immunofluorescence staining of EGFR (red) and DAPI (blue) was analyzed by confocal microscopy with z-stacks. Yellow and green lines represented corresponding points in the orthogonal planes, which confirmed distribution of the labels within the pictured cells after the summation of serial optical sections. The scale bar represents 10 μm. C, shown is EGFR overexpression in A431 cells followed by cellular fractionation. The cellular localization of EGFR was analyzed by WB. NE, nuclear extract. D, nuclear localization of EGFR in several gefitinib-resistant cell line pairs was analyzed as described in A.

Wei-Chien Huang, et al. J Biol Chem. 2011 June 10;286(23):20558-20568.
6.
FIGURE 6.

FIGURE 6. From: Nuclear Translocation of Epidermal Growth Factor Receptor by Akt-dependent Phosphorylation Enhances Breast Cancer-resistant Protein Expression in Gefitinib-resistant Cells.

Nuclear EGFR enhances transcriptional activation of BCRP/ABCG2 in A431/GR cells via recruitment to the BCRP/ABCG2 promoter. A, the DNA binding consensus sites of EGFR in BCRP/ABCG2 promoter are shown. B, three probes for DNA affinity precipitation assay were designed according to the sequence of human BCRP/ABCG2 promoter. C, the binding of EGFR to BCRP/ABCG2 promoter was analyzed by DNA affinity precipitation assay (left). The inputs of probes were shown in at the right. D, the binding of EGFR to BCRP/ABCG2 promoter was analyzed by chromatin immunoprecipitation (ChIP). E, transcriptional activities of BCRP/ABCG2 in A431 and A431/GR cells were analyzed by luciferase reporter assay. F, effects of EGFR Ser-229 mutations on EGFR-induced BCRP/ABCG2 promoter activity were analyzed in A431/GR cell by luciferase reporter assay. Error bars in E and F denote S.E. (n = 3). G, protein expression of BCRP/ABCG2 in HEK293 cells co-transfected with EGFR Ser-229 mutants. H, the proposed model for the mechanism underlying nuclear EGFR-mediated BCRP/ABCG2 expression conferring gefitinib resistance.

Wei-Chien Huang, et al. J Biol Chem. 2011 June 10;286(23):20558-20568.

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