Fig 1: hnRNPM promotes the expression of CDR1as in PDLSCs. In the RNA pull-down assay, the biotinylated CDR1as probe (biotin-CDR1as) was designed to pull down CDR1as and RBPs. Biotinylated random oligo (biotin-NC) was used as a negative control. A. Normalized CDR1as levels in input and biotin-CDR1as and biotin-NC elutions of the pull-down assay, as measured by qRT-PCR. B. Silver staining of eluted samples from the PDLSC pull-down assay. C. hnRNPM protein levels in eluted samples from the pull-down assay, as measured by Western blot. D. hnRNPM mRNA expression levels in PDLSCs transfected with si-NC and four siRNAs targeting different regions of hnRNPM, as analysed by qRT-PCR. E. hnRNPM protein expression levels in PDLSCs transfected with si-NC and four siRNAs targeting different regions of hnRNPM, as analysed by Western blot. F. CDR1as levels in PDLSCs transfected with si-NC and four siRNAs targeting different regions of hnRNPM, as analysed by qRT-PCR. Quantitative qRT-PCR data are presented as mean ± SD of three experiments. **P < .01, by Student's t test
Fig 2: RBPs were highly expressed in the FAD NCC models in vitro. (A) The validation of GEO dataset GSE3548 data by real-time PCR. The expressions of HNRNPM, HNRNPC, LARP6, and TRMT1 were significantly up-regulated in FAF-group H9-NCCs compared to control-group H9-NCCs (i). The expressions of PPIL4 and RCAN2 were significantly up-regulated in the shRFC-group H9-NCCs compared to Scramble-group H9-NCCs. (B) Western blots showed evidence of up-regulation of LARP6, hnRNPC and TRMT1 proteins in the FAF-group H9-NCCs (i) and PPIL4 and RCAN2 proteins in the shRFC-group H9-NCCs. n=3. * P<0.05, ** P<0.01. FAD, folic acid deficiency; RFC, reduced folate carrier; hnRNPM, heterogeneous nuclear ribonucleoprotein M; hnRNPC, heterogeneous nuclear ribonucleoprotein C; LARP6, La ribonucleoprotein domain family member 6; TRMT1, tRNA methyltransferase 1; PPIL4, peptidylprolyl isomerase like 4; RCAN2, regulator of calcineurin 2.
Fig 3: (A) Quantification of AS1-S RNA in RNA-immunoprecipitated samples from MDBK 3´LTR AS1-S cells. RNA was purified from the RNA-protein complexes obtained by RNA immunoprecipitation, followed by real-time RT-PCR. The results are shown as the expression levels relative to the control sample. Data are presented as the mean ± standard deviation (n = 3), and a t-test was performed for statistical analysis. P < 0.05 was defined as statistically significant. (B) Scatter plots showing the sequencing reads obtained by RNA immunoprecipitation. The X and Y axes show the read counts obtained by RIP-seq with the control MAb and anti-hnRNPM MAb, respectively. Genes that met the following criteria were defined as hnRNPM-binding RNAs and are shown as red dots: a read count >10, hnRNPM/control ratio >2.0. (C) Venn diagram showing the overlapping hnRNPM-binding genes in MDBK 3´LTR AS1-S and MDBK mock cells. (D–F) Gene ontology (GO) analysis of the 4,607 hnRNPM-binding genes detected only in MDBK 3´LTR AS1-S cells. The enriched terms in molecular function (MF) (D) and biological process (BP) (E), and the KEGG pathway enrichment analysis (F) are shown separately. The size of the bubbles indicates the gene count, and the color of the bubbles indicates the adjusted P-value. An adjusted P-value < 0.05 was defined as significant.
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