Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (AB5408)

Immunofluorescent analysis of 4% Paraformaldehyde fixed, 0.1% Triton X-100 permeabilized HeLa cells with ab5408 at 1/500 dilution followed by ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). ab206369 Anti-beta Tubulin rabbit monoclonal antibody (Alexa Fluor^® 594) was used for counterstain at a 1/100 dilution (Red). Nuclear DNA was labelled with DAPI (shown in blue)

Confocal image showing nuclear staining in HeLa cell line, the signal decreased after phosphatase treatment at 37°C for 2h.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (AB5408)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab5408 at 1/1000 dilution (0.1ug) followed by Goat anti-Mouse IgG (Alexa Fluor^® 488, ab150113) secondary at 1/2000 dilution (Red) compared with a Mouse monoclonal IgG isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

• ChIP

Lab

ChIP - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (AB5408)

Chromatin was prepared from HeLa cells according to the Abcam Dual-X-ChIP protocol. Cells were fixed with formaldehyde for 10min.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab5408 (red), or 5 µg of Mouse IgG1 ab18443 (gray) and 25 µl of Protein A/G Dynabeads. The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
Primers and probes are located in the first kb of the transcribed region.

• IP

Lab

Immunoprecipitation - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (AB5408)

RNA polymerase II CTD repeat YSPTSPS (phospho S5) was immunoprecipitated from HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab5408 at 1/50 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab5408 at 1/1000 dilution.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408) at 1/50 dilution

Lane 1:

HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg

Lane 2:

HeLa whole cell lysate

Lane 3:

Mouse IgG1, kappa monoclonal isotype control (ab184443) instead of ab5408 in HeLa whole cell lysate

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/10000 dilution

Predicted band size: 240 kDa

false

Exposure time: 10s

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (AB5408)

Immunofluorescent analysis of 4% Paraformaldehyde fixed, 0.1% Triton X-100 permeabilized NIH/3T3 cells with ab5408 at 1/500 dilution followed by ab150113 Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). ab206369 Anti-beta Tubulin rabbit monoclonal antibody (Alexa Fluor^® 594) was used for counterstain at a 1/100 dilution (Red). Nuclear DNA was labelled with DAPI (shown in blue)

Confocal image showing nuclear staining in NIH/3T3 cell line, the signal decreased after phosphatase treatment at 37°C for 2h.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (AB5408)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling RNA polymerase II CTD repeat YSPTSPS (phospho S5) with ab5408 at 1/1000 dilution (0.1ug) followed by Goat anti-Mouse IgG (Alexa Fluor^® 488, ab150113) secondary at 1/2000 dilution (Red) compared with a Mouse monoclonal IgG isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

• WB

Lab

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (AB5408)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 23071310)

All lanes:

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408) at 1/1000 dilution

Lane 1:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate (Untreated membrane) at 10 µg

Lane 2:

MCF7 whole cell lysate (Phosphatase treated membrane) at 10 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/5000 dilution

Predicted band size: 260 kDa

false

Exposure time: 3.25s

• WB

Lab

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (AB5408)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 23071310)

All lanes:

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408) at 1/1000 dilution

Lane 1:

PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate (Untreated membrane) at 10 µg

Lane 2:

PC-12 whole cell lysate (Phosphatase treated membrane) at 10 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/5000 dilution

Predicted band size: 260 kDa

false

Exposure time: 15s

• WB

Lab

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (AB5408)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 23071310)

All lanes:

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate (Untreated membrane) at 10 µg

Lane 2:

NIH/3T3 whole cell lysate (Phosphatase treated membrane) at 10 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/5000 dilution

Predicted band size: 260 kDa

false

Exposure time: 3.25s

• I-ELISA

Lab

Indirect ELISA - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (AB5408)

Indirect ELISA analysis of ab5408 at 1000-0 ng/ml. The Secondary antibody used was Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) at 1/1000 dilution.

Antigen : RNA polymerase II CTD (phospho S2) peptide, phospho S5 peptide, phospho T4 peptide, phospho Y1peptide, phospho S7 peptide, non-phospho peptide.

Antigen concentration : 100 ng/ml

• Dot

Lab

Dot Blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (AB5408)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

All lanes:

Dot Blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (ab5408) at 1/1000 dilution

Lane 1:

RNA polymerase II CTD repeat YSPTSPS (phospho S2) peptide

Lane 2:

RNA polymerase II CTD repeat YSPTSPS (phospho S5) peptide

Lane 3:

RNA polymerase II CTD repeat YSPTSPS (phospho T4) peptide

Lane 4:

RNA polymerase II CTD repeat YSPTSPS (phospho Y1) peptide

Lane 5:

RNA polymerase II CTD repeat YSPTSPS (phospho S7) peptide

Lane 6:

RNA polymerase II CTD repeat YSPTSPS non-phopho peptide

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/5000 dilution

Predicted band size: 260 kDa

false

Exposure time: 3min

• WB

CiteAb

Western blot - Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade (AB5408)

RNA polymerase II CTD repeat YSPTSPS (phospho S5) western blot using anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody [4H8] - ChIP Grade ab5408. Publication image and figure legend from DePaolo, J. S., Wang, Z., et al., 2016, Oncotarget, PubMed 27659526.

ab5408 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab5408 please see the product overview.

ARD1-dependent acetylation enhances AR transcriptional activity(A) Luciferase reporter assays in DU 145 cells co-transfected with PSA-Luc reporter with AR-WT, AR-618R, AR-618Q, or empty vector respectively. Results presented as mean of three replicates ± SD. (B and C) Differential binding of AR-WT, -618R, or -618Q to PSA and TMPRSS2 promoters by ChIP assays using anti-AR antibodies. Normalized relative densitometry was shown above. (D) Quantification of in vivo expression levels of mRNA for TMPRSS2 (AR positively-regulated) and UGT2B15 (AR negatively-regulated) in DU 145 cells stably expressing AR-WT, AR-618R, AR-618Q, or empty vector by qRT-PCR. Values represent the mean of triplicate experiments ± SD. (E) Immunoblot of co-immunoprecipitated RNA polymerase II by transiently-transfected AR-WT, AR-618R, or AR-618Q in DU 145 cells.

false