Proteins were extracted, and equal amounts of protein were separated on 5%-15% sodium dodecyl sulfate–polyacrylamide gels as previously described [20 (link)]. Separated proteins were transferred onto nitrocellulose membranes, and the blots were probed with primary antibodies (1:1,000 dilution) overnight at 4°C. Antibodies specific for the following factors were used: Pim-1 (sc-13513, Santa Cruz Biology Technology, Santa Cruz, CA), Pim-2 (CST 4730, Cell Signaling Technology, Beverly, MA), Pim-3 (CST 4165), phosphorylated (p)Bad Ser112 (CST 9296), Bad (CST 9292), p4EBP1 Ser65 (CST 9451), 4EBP1 (CST 9452), pS6K (CST 2211), S6K (CST 2217), Bcl-xL (sc-1690), Mcl-1 (sc-819), PARP (BD 556-494), caspase-3 (CST 9662), light chain 3B (LC3B; CST 3868), Beclin-1 (ab51031), pATM Ser1981 (CST 4526), ATM (sc-239-21), pChk2 Thr68 (CST 2661), Chk2 (sc-5278), pAkt Ser473 (CST 9271), Akt (CST 9272), pPRAS40 Thr246 (CST 2997), PRAS40 (CST 2691), and α-tubulin (Sigma-Aldrich). Antibody binding was detected using an enhanced chemiluminescence system according to the manufacturer’s protocol (Amersham Biosciences, Piscataway, NJ).
Protocol detail
Western Blot Analysis of Signaling Proteins
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4ebp1
Antibodies
Antibody
Beclin 1
Caspase 3
Chemiluminescence
Dilution
Light
Membranes
Nitrocellulose
Polyacrylamide gels
Proteins
Sodium dodecyl sulfate
α tubulin
Corresponding Organization :
Other organizations : Seoul National University
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Protocol cited in 1 other protocol
Variable analysis
independent variables
- None explicitly mentioned
- Pim-1 protein levels
- Pim-2 protein levels
- Pim-3 protein levels
- Phosphorylated Bad (pBad) Ser112 protein levels
- Bad protein levels
- Phosphorylated 4EBP1 (p4EBP1) Ser65 protein levels
- 4EBP1 protein levels
- Phosphorylated S6K (pS6K) protein levels
- S6K protein levels
- Bcl-xL protein levels
- Mcl-1 protein levels
- PARP protein levels
- Caspase-3 protein levels
- Light chain 3B (LC3B) protein levels
- Beclin-1 protein levels
- Phosphorylated ATM (pATM) Ser1981 protein levels
- ATM protein levels
- Phosphorylated Chk2 (pChk2) Thr68 protein levels
- Chk2 protein levels
- Phosphorylated Akt (pAkt) Ser473 protein levels
- Akt protein levels
- Phosphorylated PRAS40 (pPRAS40) Thr246 protein levels
- PRAS40 protein levels
- Equal amounts of protein were separated on 5%-15% sodium dodecyl sulfate–polyacrylamide gels
- Separated proteins were transferred onto nitrocellulose membranes
- α-tubulin protein levels
- None explicitly mentioned
- None explicitly mentioned
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