TCL and NCF, and histones were resolved on 10 and 18 % polyacrylamide SDS-PAGE, respectively, and transferred to PVDF membrane. Proteins on PVDF membrane were hybridized with anti-H3 (Upstate-06-755; 1:2000 dilution), H4 (Millipore-07-108; 1:4000 dilution), H3S10ph (Millipore-06-570; 1:7000 dilution), H4K16ac (Millipore-07-329; 1:8000 dilution), H4K20me3 (Abcam-9053; 1:4000 dilution), β-actin (Sigma-A5316; 1:10,000 dilution), MSK1 (Santacruz-9392; 1:2000 dilution), ph-MSK1 (Abcam-31190; 1:3000 dilution), ERK1/2 (Santacruz-292838; 1:2000 dilution), ph-ERK (Cell signaling-9910; 1:2000 dilution), p38 (Santacruz-728; 1:2000 dilution), ph-p38 (Cell signaling-9910; 1:2000 dilution), and anti-flag (Sigma-F3165; 1:5000 dilution). Signal was visualized using horseradish peroxidase-conjugated anti-rabbit/mouse secondary antibody and ECL plus chemiluminescence kit (Amersham). Wherever required, the densitometry analysis was done on immunoblot and membrane to determine their mean intensities using ImageJ software. For native proteins, mean intensity of immunoblot was normalized with the stained PVDF membrane; for phosphorylated forms, mean intensity of immunoblot was normalized with immunoblot of native proteins. The resulted value was used to express their mean relative levels in resection margin and tumor.
Ph msk1
Manufactured by Abcam
Ph-MSK1 is a recombinant protein that corresponds to the phosphorylated form of Mitogen and Stress Activated Protein Kinase 1 (MSK1). MSK1 is a serine/threonine protein kinase that is activated in response to various stimuli, including mitogens and stress.
Automatically generated - may contain errors
Lab products found in correlation
H4k20me3, by Abcam (1 mentions)
Ecl plus chemiluminescence kit, by Cytiva (1 mentions)
Ph p38, by Cell Signaling Technology (1 mentions)
H3s10ph, by Merck Group (1 mentions)
Pherk, by Cell Signaling Technology (1 mentions)
Anti flag, by Cell Signaling Technology (1 mentions)
H4k16ac, by Merck Group (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
β actin, by Abcam (1 mentions)
Benchmark gx, by Roche (1 mentions)
2 protocols using ph msk1
1
Immunoblot Analysis of Histone Modifications
2
Immunohistochemistry Analysis of Tumor Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Immunohistochemical analysis was carried out on 4 µm sections from each formalin fixed paraffin embedded (FFPE) tissue block which were then mounted on positively charged glass slides. One section from each block was initially stained with hematoxylin-eosin to verify that adequate amounts of tumour were present.
Antibodies used were, anti-mouse p-p38 monoclonal antibody (Cell signaling) at 1 : 100 dilution, anti-rabbit phospho-MSK1 monoclonal antibody (ph-MSK1, Abcam) at 1 : 100 dilution, and an antirabbit Ki67 monoclonal antibody (Roche-30-9, ready to use) following the manufacturer's instructions. Immunohistochemistry was performed using an automated immune-stainer (Ventana Bench-Mark GX; Ventana Inc.). FFPE sections were deparaffinised followed by cell conditioning. Antigen retrieval was carried out using a Tris-based reaction buffer (pH 7.6) prior to application of primary antibody for 30 minutes. Visualisation was performed using Avidin Biotin detection. A bladder cancer sample was used as a positive control for p-p38, p-MSK1and Ki67. Omission of primary antibody during staining served as negative controls.
Antibodies used were, anti-mouse p-p38 monoclonal antibody (Cell signaling) at 1 : 100 dilution, anti-rabbit phospho-MSK1 monoclonal antibody (ph-MSK1, Abcam) at 1 : 100 dilution, and an antirabbit Ki67 monoclonal antibody (Roche-30-9, ready to use) following the manufacturer's instructions. Immunohistochemistry was performed using an automated immune-stainer (Ventana Bench-Mark GX; Ventana Inc.). FFPE sections were deparaffinised followed by cell conditioning. Antigen retrieval was carried out using a Tris-based reaction buffer (pH 7.6) prior to application of primary antibody for 30 minutes. Visualisation was performed using Avidin Biotin detection. A bladder cancer sample was used as a positive control for p-p38, p-MSK1and Ki67. Omission of primary antibody during staining served as negative controls.
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