Component of the cleavage factor Im complex (CFIm) that plays a key role in pre-mRNA 3'-processing. Involved in association with NUDT21/CPSF5 in pre-MRNA 3'-end poly(A) site cleavage and poly(A) addition. CPSF6 binds to cleavage and polyadenylation RNA substrates and promotes RNA looping.
Belongs to the RRM CPSF6/7 family. Contains 1 RRM (RNA recognition motif) domain.
Phosphorylated upon DNA damage, probably by ATM or ATR.
Nucleus. In punctate subnuclear structures localized adjacent to nuclear speckles, called paraspeckles.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma (left) and mouse renal cell carcinoma (right) tissues labelling CPSF6 with ab99347 at 1/200 (1µg/ml). Detection: DAB.
ICC/IF image of ab99347 stained MCF7 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab99347, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
Western blot - Anti-CPSF6 antibody (ab99347)
All lanes : Anti-CPSF6 antibody (ab99347) at 0.04 µg/ml
Lane 1 : HeLa whole cell lysate at 50 µg Lane 2 : HeLa whole cell lysate at 15 µg Lane 3 : HeLa whole cell lysate at 5 µg Lane 4 : 293T whole cell lysate at 50 µg Lane 5 : NIH 3T3 whole cell lysate at 50 µg
Detection of CPSF6 by Western Blot of Immunprecipitate.
ab99347 at 1µg/ml staining CPSF6 in HeLa whole cell lysate immunoprecipitated using ab99347 at 3µg/mg lysate (1 mg/IP; 20% of IP loaded/lane).
Detection: Chemiluminescence with exposure time of 1 seconds.
IHC image of CPSF6 staining in human breast fibroadenoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab99347, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.