Fig. 3: HuR controls GAC and KGA mRNA levels in breast cancer cell lines and binds to GLS mRNA at multiple sites.
a Relative quantification of BT549 mRNA levels following doxycycline-inducible silencing of ELAVL1, using qPCR for two distinct shRNA sequences. b HuR was immunoprecipitated (western blot above, arrows indicate HuR and IgG heavy chain). The control IgG were used from rabbit, in opposing to the mouse anti-HuR antibody. Quantification of mRNA derived from 3′UTR KGA regions immunoprecipitated bound in HuR from (c) PC-3 cells using agarose gel and (d) from BT549 using qRT-PCR. Quantification of mRNA derived from 3′UTR GAC regions immunoprecipitated bound in HuR from (e) PC-3 cells using agarose gel and (f) from BT549 using qRT-PCR. Agarose gel from PC-3 (g) and q-RT-PCR from BT549 (h) revealed that HuR binds to its mRNA (as already published elsewhere126) in addition to GLS intron 14. i In vitro FRAP analysis of recombinant mKO2-HuR incubated with in vitro transcribed control RNA or intron 14. The bar plot (right) denotes T1/2 recovery times. Statistical significance derived from Two-sided Welch’s t-test (a, d, f, h, and i), error bars are SEM; qRT-PCR assays were evaluated in triplicate; otherwise, each point represents a replicate. *p < 0.05, **p < 0.01, ***p < 0.0001.
