Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human breast carcinoma tissue sections labeling RPS6 with ab81081 at 1/50 dilution (3.86 µg/ml). Heat mediated antigen retrieval with sodium citrate buffer (pH 6.0, epitope retrieval solution 1) for 10mins. Goat Anti-Rabbit & Mouse IgG (HRP) was used as the secondary antibody. Hematoxylin was used as a counterstain. Cytoplasmic staining on human breast carcinoma without lambda phosphatase treatment (image A); no signal can be observed after treatment with lambda phosphatase (image B), performed on a Leica Biosystems BOND™ RX instrument.
The section was incubated with ab81082 for 30 mins at room temperature.

Immunocytochemistry/ Immunofluorescence analysis of MCF7 (human breast adenocarcinoma epithelial cell) treated with lamda phosphatase cells labeling RPS6 with purified ab81081 at 1/500 (1.3 µg/ml). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/ml) dilution. DAPI was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Flow cytometry analysis of MCF7 grown in serum-free media overnight, then grown in 20% FBS media for 30 minutes labeling RPS6 with purified ab81081 at 1/640 dilution (1.00 µg/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as a secondary antibody. Isotype control -Rabbit monoclonal IgG (ab172730) / Black. Unlableled control -Unlabelled cells / blue.Untreated cells (Green).

Formalin-fixed, paraffin-embedded human breast carcinoma tissue stained for RPS6 (phospho S240 + S244) using ab81081 at 1/50 dilution in immunohistochemical analysis.

Dot blot analysis of RPS6 (phospho S240 + S244) peptide (Lane 1), RPS6 (phospho S240) peptide (Lane 2), RPS6 (phospho S244) peptide (Lane 3) and RPS6 non-phospho peptide (Lane 4) RPS6 labeling (phospho S240 + S244) with purified ab81081 at 0.643 µg/mL. ab97051 (Peroxidase conjugated goat anti-rabbit IgG (H+L)) was used as the secondary antibody at 0.01 µg/mL.

Blocking and dilution buffer: 5% NFDM/TBST.

ELISA antigen dose-response curve using purified ab81081 at 0.1-1000 ng/mL. Antigen concentration of 0-10000 ng/mL. A Alkaline Phosphatase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch) (1/2500) was used as the secondary antibody.

RPS (phospho S240 + S244): RA-S(p)-TSK-S(p)-ESSQKC
RPS (phospho S240) : RA-S(p)-TSKSESSQKC
RPS (phospho S244): RASTSK-S(p)-ESSQKC
RPS non-phospho: RASTSKSESSQKC