anti cpsf6  (Proteintech)

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: CPSF6 immunocomplexes were specifically enriched using a validated rabbit polyclonal antibody (Proteintech 15489‐1‐AP), with rabbit IgG (Millipore 12–370) serving as a negative control.

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: CPSF6 immunocomplexes were specifically enriched using a validated rabbit polyclonal antibody (Proteintech 15489‐1‐AP), with rabbit IgG (Millipore 12–370) serving as a negative control.

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: CPSF6 immunocomplexes were specifically enriched using a validated rabbit polyclonal antibody (Proteintech 15489‐1‐AP), with rabbit IgG (Millipore 12–370) serving as a negative control.

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: CPSF6 immunocomplexes were specifically enriched using a validated rabbit polyclonal antibody (Proteintech 15489‐1‐AP), with rabbit IgG (Millipore 12–370) serving as a negative control.

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