Fig 1: The transcriptional function of CBFB is critical for breast tumor suppression. a A Venn diagram showing genes (identified by RNAseq) co-regulated by CBFB and RUNX1. b Kaplan-Meier curve showing 10-gene signature upregulated by CBFB and RUNX1 predicts disease-free survival of breast cancer patients. c IB showing the upregulation of Notch intracellular domain (NICD) of NOTCH3 in CBFB_KO and RUNX1_KO cells. d ChIPseq of RUNX1 showing the binding of RUNX1 on the NOTCH3 locus. e Anchorage independent growth assays of double knockout (DKO) of CBFB (C) and NOTCH3 (N) or DKO of RUNX1(R) and NOTCH3(N). n = 3 (biological); two asterisks, p value < 0.01 (single KO vs. double KO comparisons, two-tailed t test). f Representative IHC images showing the protein levels of CBFB, RUNX1, and NOTCH3 in human breast tumors and normal tissues. g IB showing the effect of exogenous expression of CBFB on RUNX1 and NOTCH3 expression in MCF7 and BT474 cells. h Weight and (i) volume of orthotopically transplanted tumors from MCF7 cells that were transduced with an empty vector (EV) or a lentiviral vector expressing CBFB (CBFB). Tumor incidence is shown on the top
Fig 2: CBFB and hnRNPK regulate translation. a Flow chart showing the in vitro translation assay to test the effect of a translation element (RNA) and a protein. In vitro transcribed RNA was used in the in vitro translation assays using HeLa cells lysate. b In vitro translation assay using the 1-Step Human coupled IVT kit showing the effect of CBFB and hnRNPK binding sites (T14) on RUNX1 translation. c In vitro translation assays using the 1-Step Human coupled IVT showing effect of recombinant CBFB (rCBFB) and hnRNPK (rhnRNPK) on RUNX1 translation. d In vitro translation assay using WT and CBFB_KO HEK 293 T cells lysate. See “Methods” section for details of preparation of 293 T cell lysate. e In vitro translation assay. Un-transfected (control) and hnRNPK knockdown (KD) HEK 293T cells lysate replaced the HeLa cell lysate in the 1-Step Human coupled IVT kit with or without the supplement of rhnRNPK. f IB showing the effect of CBFB deletion on the proteins encoded by bound transcripts. g IB showing the effect of hnRNPK KD on the proteins encoded by bound transcripts
Fig 3: CBFB interacts with and facilitates mRNA binding of eIF4B. a FLAG IP followed by IB showing the interaction of translation initiation factors with CBFB. 10 µg/ml RNase A was added in the co-IP lysate. b Endogenous co-IP followed by IB showing interaction of CBFB, hnRNPK, and eIF4B. 10 µg/ml RNase A was added in the co-IP lysate. c In vitro translation assays showing the effect of rCBFB, rhnRNPK, and reIF4B on RUNX1 translation in a cap-dependent or -independent manner. d RIP followed by real-time PCR to show the effect of CBFB deletion on the binding of eIF4B on CBFB-bound transcripts. Error bars are SEM, n = 3 (biological); n.s., p value > 0.05 (RIP of eIF4B in WT vs. CBFB KO cells). e A model showing the role of CBFB in translation regulation through hnRNPK and eIF4B
Fig 4: CBFB binds to RUNX1 mRNA via hnRNPK. a Silver staining of FLAG pulldown using CBFB KO MCF10A cells expressing empty vector and N-terminally FLAG tagged CBFB. b IB validation of CBFB and hnRNPK interaction. c IB showing the effect of hnRNPK knockdown on RUNX1 protein. d RNA immunoprecipitation (RIP) with CBFB antibody in WT and CBFB_KO MCF10A cells. Error bars are SEM, n = 3 (biological); two asterisks, p value < 0.01. (RIP of CBFB in WT vs. in CBFB KO cells, two-tailed t test). e RIP with hnRNPK antibody. Error bars are SEM, n = 3 (biological); two asterisks, p value < 0.01 (RIP of hnRNPK in WT vs. CBFB KO cells, two-tailed t test). f RNA pulldown assays (RPA) determining the hnRNPK-bound region within RUNX1 mRNA. F1-4, fragment 1 to 4 of 3' UTR of RUNX1 mRNA. See Methods for details. Numbers indicate the nucleotide positions. g Re-analyses of two public eCLIP datasets (GSM2423241 and GSM2423242) of hnRNPK on the RUNX1 locus. Nucleotide sequences of two poly-C tracts within the binding site of hnRNPK were shown. F3, fragment 3 of 3'-UTR; T14, truncation 14 of 3'-UTR F3; Mu1, mutation 1; Mu2, mutation 2. h RPA using recombinant CBFB in the absence or presence of recombinant hnRPNK. i RPA using recombinant hnRNPK in the absence or presence of recombinant CBFB
Fig 5: Genome-wide binding of CBFB and hnRNPK to mRNAs. RIPseq of FLAG-CBFB (a) and hnRNPK (b) in MCF10A cells. Shown are mean adjusted FPKM + 1 (log2). Red dots show the transcripts that are enriched in FLAG-CBFB RIP more than 4-fold. The rest transcripts are shown as blue dots. c A Venn diagram showing transcripts bound by both CBFB and hnRNPK. d Validation of binding of CBFB and hnRNPK to 16 selected transcripts using RIP followed by real-time PCR. Error bars are SEM, n = 3 (biological). e The top-ranked gapped motif, which was represented in 86% of the hnRNPK binding sites occurred in hnRNPK-bound transcripts
Supplier Page from Abcam for Recombinant Human CBFb protein