Fig. 2: Identification of molecular targets of HuD. | Cell Death & Disease

Fig. 2: Identification of molecular targets of HuD.

From: Loss of RNA binding protein HuD facilitates the production of the senescence-associated secretory phenotype

Fig. 2

A The mRNAs that interact with HuD were isolated via RNA immunoprecipitation and analyzed via gene expression profiling using the NanoString nCounter® Inflammation Panel. Enriched mRNAs in HuD IP (>2-fold) compared to control IgG IP are listed in the table and senescence-associated secretory phenotype (SASP) mRNAs are shown in bold. B N2a cells were transiently transfected with control siRNA or HuD siRNA and the secretory proteins in the culture media were analyzed by western blotting using the Proteome Profiler Mouse XL cytokine array. The differential expression analysis of secretory proteins revealed six proteins, including CCL2, CCL20, CXCL2, IL-6, CD40, and ENDOGLIN, which were identified as upregulated ones in the media from HuD knockdown cells. C Four candidates that not only bind to HuD but also are upregulated by HuD knockdown were determined as putative targets of HuD; Ccl2, Ccl20, Cxcl2, and Il-6. D Interaction between HuD and its putative targets was validated by RNA IP followed by RT-qPCR using anti-HuD and control IgG antibodies. Gapdh mRNA was used for normalization. Data indicate the mean ± SEM from three independent analyses. The statistical significance of the data was analyzed via Student’s t-test; *p < 0.05.

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