Figure 2
hnRNPK and active ERKs post-transcriptionally regulate p65BTK expression. (a) In vitro translation assay performed with the following plasmids: empty vector (empty); p65FL (wt), p65_msATG1, p65_nsATG1, p65_nsATG2, p77_5′UTR or p77_msATG1. +cnt indicates the positive control included in the commercial kit used for the reaction. (b) p65BTK mRNA expression in matched samples of tumoural and peritumoural colon tissue from CRC patients (same patients as in Figure 1b). mRNA was quantified by Taqman assay and expression levels normalized to phosphoglycerate kinase. (c) Western blot of 293T cells transfected with empty vector (empty) or the following plasmids: p65FL, p65_5′UTRΔK1, p65_5′UTRΔK2, p65_5′UTRΔK3, p65_5′UTRΔK4. Deletion of all four binding sites allowed p65BTK overexpression most likely by rendering the transcript as it would be a CDS. (d) Western blot of p65BTK levels in colon cancer cell lines after siRNA-mediated depletion of hnRNPK (K). Transfection with siRNAs targeting luciferase (luc) was used as a control. On the right, the percentage of hnRNPK and p65BTK protein expression of each sample as calculated and normalized to actin by ImageJ program (http://imagej.nih.gov/ij/). (e, top) Anti-hnRNPK and anti-phospho-hnRNPK western blots after RNA immunoprecipitation using anti-hnRNPK and isotype-matched control (Ig mouse) antibodies. (e, bottom) Real-time PCR of p65BTK mRNA recovered by RIP in hnRNPK and IgG immunoprecipitates. (f) Western blot of p65BTK expression and hnRNPK-Ser284 phosphorylation following ERK1/2 inhibition with the MEK1/2 inhibitor CI-1040 (10 μM). Levels of total and phospho-ERKs are also shown. Cell lysates were obtained 24 h after CI-1040 addition but for HCT116p53KO cells, where p65BTK reduction is most prominent, at 16 h. On the right, the percentage of p-hnRNPK and p65BTK protein expression of each sample was calculated and normalized to actin by ImageJ program.
