Cells were serum starved for 16 hours in Roswell Park Memorial Institute medium (RPMI) with 0.7% fetal calf serum (FCS) before inhibitor treatment. Cell lysates in RIPA buffer (50 mM Tris, pH 8, 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.5% sodium deoxylate, 1% nonyl phenoxypolyethoxylethanol [NP40]) were subjected to western blotting with the following antibodies, all from Cell Signaling Technologies (Danvers, MA, USA): pIGF-1R (3021) (phosphorylated insulin like growth factor-1-receptor); pJAK2 (3771) (phosphorylated janus kinase 2); pSTAT5 (9351) (phosphorylated signal transducer and activator of transcription 5); pMEK (9121) (phosphorylated MAPK/ERK kinase); pERK (4370) (phosphorylated extracellular signal regulated kinase); pAKT (9275) (phosphorylated protein kinase B); IGF-1R (3027); JAK2 (3230); STAT5 (9352); MEK (9122); ERK (9102); AKT (9272). Blots were stripped and reprobed against beta actin (Sigma A5441) to ensure equal loading. Immune complexes were detected using chemiluminescence (K12-045-D20, Advansta, San Jose, CA, USA).
Pmek 9121
Manufactured by Cell Signaling Technology
Sourced in United States
PMEK (9121) is a primary antibody that specifically recognizes phosphorylated forms of MEK1/2. It can be used to detect the phosphorylation status of MEK1 and MEK2 in various applications.
Automatically generated - may contain errors
Lab products found in correlation
P erk 4370, by Cell Signaling Technology (1 mentions)
Stat5, by Cell Signaling Technology (1 mentions)
Pstat5 9351, by Cell Signaling Technology (1 mentions)
Pakt 9275, by Cell Signaling Technology (1 mentions)
Igf 1r, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Avantor (1 mentions)
Rpn2232, by Cytiva (1 mentions)
β tubulin 2128, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg sc2004, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
2 protocols using pmek 9121
1
Serum Starvation and Kinase Signaling
2
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Proteins were extracted by RIPA lysis buffer (P0013B, Beyotime Institute of Biotechnology, Beijing, China) with protease inhibitor cocktails (M221, Amresco, Solon, OH, USA). Proteins were separated by electrophoresis and after electronic transfer, the membranes were blocked and incubated with specific antibodies overnight at 4 °C. Antibodies against S6K1 (2708), p-S6K1 (T389) (9234), rpS6 (2217), p-rpS6 (S235/6), p-Erk1/2 (9101), p-MEK (9121), p-Akt (S473) (9271), Akt (9272) and β-tubulin (2128) were all rabbit antibodies and purchased from Cell Signaling Technology. Horseradish peroxidase-conjugated goat anti-rabbit IgG (SC2004, Santa Cruz Biotechnology, Dallas, TX, USA) were then used to detect proteins through enhanced chemiluminescence (RPN2232, Amersham, Washington, NY, USA).
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