Journal: Oncogene

Article Title: SENP1-mediated deSUMOylation of YBX1 promotes colorectal cancer development through the SENP1-YBX1-AKT signaling axis.

doi: 10.1038/s41388-025-03302-6

Figure Lengend Snippet: Fig. 3 K26 SUMOylation-defective mutation of YBX1 significantly enhanced YBX1 binding with DDX5 protein. A Effects of YBX1 and its mutants on AKT signaling in DLD-1 cells transfected with indicated plasmids. B YBX1-binding proteins were enriched from total lysates of HEK293T cells with over-expression of Flag-YBX1 with anti-Flag magnetic beads, separated by SDS-PAGE and stained by Coomassie brilliant blue. The specific binding protein bands (shown as arrows) were subjected to MS/MS analysis. C The MS/MS identification parameters of YBX1 and its eight candidate binding partners, including IGF2BP1, HNRPU, IGF2BP3, DDX5, G3BP1, YTHDF3, IGF2BP2 and ENOA proteins. D Interaction of YBX1 with its eight candidate binding partners (GF2BP1, HNRPU, IGF2BP3, DDX5, G3BP1, YTHDF3, IGF2BP2 and ENOA) was validated in HEK293T cells transfected with indicated plasmids. E Interaction of YBX1K26R or YBX14KR with IGF2BP1, HNRPU, IGF2BP3, DDX5, G3BP1, YTHDF3, IGF2BP2 was compared to that of the wild-type YBX1 in HEK293T cells transfected with indicated plasmids. F The interaction between DDX5 with YBX1, YBX1K26R, YBX14KR was measured in CRC cells transfected with indicated plasmids. G Flag-YBX1 or Flag-YBX1K26R

Article Snippet: The following antibodies were used in this study: β-actin antibody (TA-09, Zsbio), β-tubulin antibody (TA-10, Zsbio), SUMO1 antibody (ET1606-53, Huabio), UBC9 antibody (ET1610-21, Huabio), SENP1 antibody (ET7108-53, Huabio), YBX1 antibody (ET1609-10, Huabio), DDX5 antibody (ET1705-32, Huabio), HNRPU antibody (ET7107-10, Huabio), G3BP1 antibody (HA721314, Huabio), ENOA antibody (ET1705-56, Huabio), FLAG-tag antibody (0912-1 or HA601080, Huabio), Myc-tag antibody (EM31105, Huabio), HA-tag antibody (ET1611-49, Huabio), Lamin B1 antibody (ET1606-27, Huabio), His-tag antibody (66005-1-Ig, Proteintech), IGF2BP1 antibody (22803-1-AP, Proteintech), IGF2BP2 antibody (11601-1-AP, Proteintech), IGF2BP3 antibody (14642-1-AP, Proteintech), YTHDF3 antibody (25537-1-AP, Proteintech), p-AKT (ser473) antibody (381555, ZENbioscience), AKT1 antibody (380617, ZEN-bioscience), mouse IgG (A7028, beyotime), rabbit IgG (A7016, beyotime), goat anti-rabbit IgG (HRP) (ZB2301, Zsbio), goat anti-mouse IgG (HRP) (ZB-2305, Zsbio), and anti-mouse Alexa Fluor 488 (Ab150113, Abcam).

Techniques: Mutagenesis, Binding Assay, Transfection, Over Expression, Magnetic Beads, SDS Page, Staining, Tandem Mass Spectroscopy

Journal: Clinical and Translational Medicine

Article Title: IGF2BP2 binding to CPSF6 facilitates m6A‐mediated alternative polyadenylation of PUM2 and promotes malignant progression in ovarian cancer

doi: 10.1002/ctm2.70388

Figure Lengend Snippet: The APA factor CPSF6 interacts with the m6A regulator IGF2BP1/2/3 complex in OC cells. (A) CPSF6 was related to m6A modification according to the RNA‒protein pulldown assay. (B) The binding proteins of CPSF6 were detected by IP/MS in OVCAR3 cells. (C) List of the top 10 proteins for CPSF6‐IP. (D) IP assay using a CPSF6 antibody with or without RNaseA to detect the IGF2BP1, IGF2BP2 and IGF2BP3 proteins in OC cells. (E) IP assay of IGF2BP1/2/3 with or without RNaseA to detect CPSF6. (F) The distribution of proteins was detected by nucleocytoplasmic separation. (G) The colocalization of the IGF2BP1/2/3 complex and CPSF6 was detected by an immunofluorescence assay.

Article Snippet: Target antigens were probed with primary antibodies against CPSF6 (Santa Cruz Biotechnology, sc‐100692) and IGF2BP family members (Proteintech: IGF2BP1 22803‐1‐AP; IGF2BP2 11601‐1‐AP; IGF2BP3 14642‐1‐AP) through overnight incubation at 4°C.

Techniques: Modification, Binding Assay, Protein-Protein interactions, Immunofluorescence

Journal: Clinical and Translational Medicine

Article Title: IGF2BP2 binding to CPSF6 facilitates m6A‐mediated alternative polyadenylation of PUM2 and promotes malignant progression in ovarian cancer

doi: 10.1002/ctm2.70388

Figure Lengend Snippet: CPSF6 RS domain directly binds to the C‐terminal KH domains of IGF2BP2. (A) Co‐IP assay of Flag‐CPSF6 truncated and Myc‐IGF2BP1/2/3 in 293T cells. (B, C) Schematic diagram of CPSF6 truncated (B) and IGF2BP2 truncated (C) proteins. (D) Western blot analysis of GST‐CPSF6‐RS or His‐IGF2BP1/2/3 proteins of GST‐input and pulldown samples. (E) Co‐IP assay of Myc‐IGF2BP2 truncated and Flag‐CPSF6 in 293T cells. (F) The structure and site of the CPSF6 RS‐IGF2BP2 interaction complex were predicted by AlphaFold3.

Article Snippet: Target antigens were probed with primary antibodies against CPSF6 (Santa Cruz Biotechnology, sc‐100692) and IGF2BP family members (Proteintech: IGF2BP1 22803‐1‐AP; IGF2BP2 11601‐1‐AP; IGF2BP3 14642‐1‐AP) through overnight incubation at 4°C.

Techniques: Co-Immunoprecipitation Assay, Western Blot

Journal: Clinical and Translational Medicine

Article Title: IGF2BP2 binding to CPSF6 facilitates m6A‐mediated alternative polyadenylation of PUM2 and promotes malignant progression in ovarian cancer

doi: 10.1002/ctm2.70388

Figure Lengend Snippet: Joint analysis of multiple sequencing for screening target genes in OVCAR3 cells. (A, B) PAS‐seq analysis of IGF2BP2 knockdown in OVCAR3, the volcano map of long and short genes (A), and the proximal and distal PAS uses a S‐shaped curve (B). (C) The PAS distribution of short and long APA genes after IGF2BP2 knockdown. (D) The volcano plot of IGF2BP2 binding genes by RIP‐seq. (E) Overlap of the CPSF6 binding target genes. (F) The enrichment motif analysis of CPSF6 eCLIP‐seq. (G) Venn diagram showing the overlap of the multiple sequencing results. (H) The peak map of CPSF6 binding sites. (I) Joint analysis of m6A‐seq and CPSF6 eCLIP‐seq.

Article Snippet: Target antigens were probed with primary antibodies against CPSF6 (Santa Cruz Biotechnology, sc‐100692) and IGF2BP family members (Proteintech: IGF2BP1 22803‐1‐AP; IGF2BP2 11601‐1‐AP; IGF2BP3 14642‐1‐AP) through overnight incubation at 4°C.

Techniques: Sequencing, Knockdown, Binding Assay

Journal: Clinical and Translational Medicine

Article Title: IGF2BP2 binding to CPSF6 facilitates m6A‐mediated alternative polyadenylation of PUM2 and promotes malignant progression in ovarian cancer

doi: 10.1002/ctm2.70388

Figure Lengend Snippet: METTL3 and IGF2BP2 regulate the APA process to affect the stability of PUM2 with m6A‐dependent and CPSF6‐binding manner in OC cells. (A, B) The expression ratio of long and short transcripts of target genes after knockdown of METTL3 (A) and IGF2BP2 (B) in OC cells, respectively. (C–F) The interaction between 3′UTR of PUM2 mRNA and CPSF6 (C), METTL3 (D), IGF2BP2 (E), and m6A (F) antibodies was validated by using eCLIP‐seq, RIP‐PCR and meRIP‐seq. (G) Distribution of the CPSF6 binding sites across PUM2 mRNA transcript as identified by eCLIP‐seq. (H) Distribution of m6A peaks across PUM2 mRNA as identified by meRIP‐seq. (I) IGV tracks showing the enrichment of PUM2 mRNA according to PAS‐seq after METTL3 and IGF2BP2 knockdown in OVCAR3 cells. (J) The abundance of PUM2 long and short transcripts was identified by 3′RACE assay in OC cells with METTL3 or IGF2BP2 knockdown. (K) The expression of PUM2 was detected by western blot after knocking down METTL3 and IGF2BP2 in OC cells. (L) Decay curves and half‐life (T1/2) of PUM2 mRNA in OVCAR3 cells with METTL3 and IGF2BP2 knockdown were derived from mRNA stability profiling. (M) The interaction between CPSF6 and PUM2 mRNA was detected by RIP‐qPCR following METTL3 and IGF2BP2 knockdown in OVCAR3 cells. (N) Scheme of m6A sites WT and MUT nucleotide sequence surrounding PUM2 pPAS in 3′UTR, and dual luciferase reporter assay performed by cotransfecting PUM2 3′UTR WT or MUT and IGF2BP2 plasmid.

Article Snippet: Target antigens were probed with primary antibodies against CPSF6 (Santa Cruz Biotechnology, sc‐100692) and IGF2BP family members (Proteintech: IGF2BP1 22803‐1‐AP; IGF2BP2 11601‐1‐AP; IGF2BP3 14642‐1‐AP) through overnight incubation at 4°C.

Techniques: Binding Assay, Expressing, Knockdown, Western Blot, Derivative Assay, Sequencing, Luciferase, Reporter Assay, Plasmid Preparation

Journal: Cancer Cell International

Article Title: Stabilization of UCA1 by N6-methyladenosine RNA methylation modification promotes colorectal cancer progression

doi: 10.1186/s12935-021-02288-x

Figure Lengend Snippet: IGF2BP2 serves as a reader for m6A modified UCA1. A The workflow of in vivo S1m-tagging RNA pulldown assay. B Detection of the YTHDF1, YTHDF2 and YTHDF3 by western blot, after in vivo RNA pulldown. C Detection of the IGF2BP1, IGF2BP2 and IGF2BP3 by western blot, after in vivo RNA pulldown. D The interaction between UCA1 and IGF2BP2 was confirmed by RIP assay. Error bars represent SD, n = 3, ***P < 0.005. E Mutation on m6A motif of UCA1 impairs the interaction of IGF2BP2 with UCA1. Left, the schematic of the A to C mutation on UCA1 m6A motif; right, the representative western blot showing that the mutant UCA1revealed little interaction with IGF2BP2 and UCA1, in contrast to WT UCA1

Article Snippet: WTAP (56501) and METTL3 (96391) were purchased from Cell Signaling Technology (Danvers, MA), YTHDF1 (17479-1-AP), YTHDF2 (24744-1-AP), YTHDF3 (25537-1-AP), IGF2BP1 (22803-1-A), IGF2BP2 (11601-1-AP), IGF2BP3 (14642-1-AP) were from Proteintech (Chicago, IL, USA).

Techniques: Modification, In Vivo, Western Blot, Mutagenesis