Ab32413, at a 1/100 dilution, staining RSK1 p90 in paraffin embedded human cervical carcinoma tissue by Immunohistochemistry.
Left image : Untreated.
Right image : Treated with Phosphatase.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

All lanes : Anti-RSK1 p90 (phospho T359 + S363) antibody [E238] (ab32413) at 1/1000 dilution (purified)

Lane 1 : NIH/3T3 (mouse embryonic fibroblast) grown in serum free media for 4 hour swhole cell lysate
Lane 2 : NIH/3T3 grown in serum free media for 4 hours, then treated with 20% FBS for 30minutes whole cell lysate
Lane 3 : NIH/3T3 grown in serum free media for 4 hours, then treated with 20% FBS for 30 minutes whole cell lysate. Then the membrane was incubated with alkaline phosphatase

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 90 kDa
Observed band size: 90 kDa

Blocking/Diluting Buffer and concentration 5% NFDM/TBST

Purified ab32413 at 1/100 dilution (2µg) immunoprecipitating RSK1 p90 in NIH/3T3 whole cell lysate.
Lane 1 (input): NIH/3T3 (Mouse embryonic fibroblast) serum starved for 4h, then treated with 20% FBS for 30min whole cell lysate 10µg
Lane 2 (+): ab32413 + NIH/3T3 whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab171376 in NIH/3T3 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 90 kDa
Lower bands could be cleavage product. (PMID: 29290690)

Dot blot analysis of RSK1 p90 (phospho T359 + S363) phospho peptide (Lane 1), RSK1 p90 phospho T359 phospho peptide (Lane 2), RSK1 p90 phospho S36 phospho peptide (Lane 3) and RSK1 p90 non-phospho peptide (Lane 4) labeling RSK1 p90 (phospho T359 + S363) phospho peptide with purified ab32413 at a dilution of 1/1000 dilution (2.056ug/ml). A Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) was used as the secondary antibody at a dilution of 1/20,000 dilution.

Blocking buffer: 5% NFDM/TBST. Dilution buffer: 5% NFDM /TBST .

BT549 cells were incubated at 37 °C for 1h with vehicle control (0 μM) and different concentrations of PD 173074 (ab141117) in DMSO. Decreased expression of RSK1 p90 (phospho T359 + S363) (ab32413) correlates with an increase in PD 173074 concentration, as described in literature.

Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 10 μg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with ab32413 at 1/5000 dilutionand ab8227 at 1 μg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.

All lanes : Anti-RSK1 p90 (phospho T359 + S363) antibody [E238] (ab32413) at 1/20000 dilution

Lane 1 : A431 cell lysate untreated.
Lane 2 : A431 cell lysate treated with EGF.

Predicted band size: 90 kDa
Observed band size: 90 kDa