Following the experimental treatment, Western blot analysis were performed as previously described (6 (link)). Immunoblot analyses were carried out on lysates extracted from cells or tumors. Protein concentration was determined, and equal amounts of total proteins were separated on SDS-PAGE. Antibodies used included total (SCT #2602) and phospho-Pak1(SC #2606), Mek (SCT#9121), phospho-Mek pSer298(SCT#9128), Erk (SCT#9102), phospho-Erk1/2 (pThr202/pTyr204) (SCT#9101), phospho-ATM, phospho-ATR, phospho-Chk1, phospho-Chk2, phospho-p53, p21Cip SCT DNA damage sampler kit, #9947), cyclin B(SCT#4128), cyclin D1(SCT#2978), GAPDH (SCT#4138) were from Cell Signaling Technology.
Protocol detail
Western Blot Analysis of Cell Signaling
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Antibodies
Cyclin b
Cyclin d1
Dna damage
Erk1
Experimental treatment
Gapdh
Immunoblot analyses
Pak1
Proteins
Sds page
Tumors
Western blot analysis
Corresponding Organization :
Other organizations : Fox Chase Cancer Center
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Variable analysis
independent variables
- Experimental treatment
- Protein levels (total and phosphorylated forms) of Pak1, Mek, Erk, ATM, ATR, Chk1, Chk2, p53, p21Cip, cyclin B, cyclin D1
- GAPDH levels (used as a loading control)
- Protein concentration (equal amounts of total proteins were separated on SDS-PAGE)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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