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. 2014 Oct 25;12:59. doi: 10.1186/s12964-014-0059-5

Figure 3.

Figure 3

CLDN4 knockdown affects the phosphorylation and kinase activity of EphA2. (A) Representative Western blot showing the effect of CLDN4 knockdown and re-expression on EphA2 Ser897 phosphorylation. (B) Histogram showing the mean ± SEM level of pEphA2-Ser897 determined from 6 independent experiments expressed as the fold change relative to that in the scrambled siRNA control 2008/SCB cells after normalization to β-actin. * p < 0.05 versus 2008/SCB. (C) Measurement of EphA2 kinase activity in 2008/SCB and CLDN4KD cells using a human phospho-receptor tyrosine kinase array containing 49 different kinase substrates that include EphA1-5, EphA10 and EphB1-4, 6. (D) Quantification of the relative level of EphA2 tyrosine phosphorylation by densitometry. Results are presented as mean ± SEM (n = 3). * p < 0.05 versus 2008/SCB. (E) Detection of phosphotyrosine in immunoprecipitates prepared by using anti-EphA2 antibodies. Immunoprecipitates were probed with pan anti-phosphotyrosine antibody conjugated to horseradish peroxidase (HRP). (F) Histogram showing the average level of phosphotyrosine determined from 3 independent experiments expressed as the fold change relative to that in the scrambled siRNA control 2008/SCB cells after normalization to total EphA2. Results are presented as mean ± SEM (n = 3). * p < 0.05 versus 2008/SCB.