Skip to main content
. Author manuscript; available in PMC: 2012 Nov 30.
Published in final edited form as: Oncogene. 2011 Oct 3;31(22):2761–2772. doi: 10.1038/onc.2011.452

Figure 5. BAG-1 regulation of EGFR expression and function is dependent on NF-κB1 expression.

Figure 5

(A) (i) HCT116 cells were transfected with control negative / BAG-1 siRNA or control plasmid / BAG-1L expression plasmid for 48 hours. Q-RT-PCR was carried out to show EGFR mRNA abundance; all mRNA values were normalised to the housekeeping gene, TBP. Data is presented as a percentage change of levels in control transfected cells, represented by the dotted line. Data points are of three independent experiments carried out in triplicate (± standard deviation). Statistical analysis was carried out using a Student’s t-test * = P < 0.05; ** = P < 0.01; *** = P < 0.001. (ii) Western analysis to show regulation of EGFR protein +/− BAG-1 expression. Equal loading was confirmed by α-tubulin. Data is representative of three independent experiments. (B) HT29 cells (positive for COX-2 expression) were transfected with control negative or BAG-1 siRNA for 48 hours. Q-RT-PCR was carried out to show PTGS2 and EGFR mRNA abundance; all mRNA values were normalised to the housekeeping gene, TBP. Data is presented as a percentage change of levels in control transfected cells, represented by the dotted line. Data points are of three independent experiments carried out in triplicate (± standard deviation). Statistical analysis was carried out using a Student’s t-test * = P < 0.05; ** = P < 0.01; *** = P < 0.001. (C) NF-κB1+/+ and NF-κB1−/− MEF cells were transfected with 25nM of control siRNA or mBAG-1 siRNA. Western analysis was carried out to show the regulation of EGFR protein expression following suppression of the BAG-1 protein in NF-κB1+/+ and −/− MEF cells. Equal loading was confirmed by α-tubulin. Results are representative of three independent experiments. (D) HCT116 cells treated with EGF (10ng/ml) for 72 hr after siRNA transfection either in the presence or absence of an EGFR inhibitor CL-387.785 (10μM) added 1h prior to EGF treatment. Activation of the EGFR upon EGF treatment was shown by the phosphorylation status of the receptor, specificity confirmed by the blockade with the EGFR inhibitor. Western blotting was used to confirm suppression of BAG-1 expression by siRNA. Equal loading was confirmed by α-tubulin.