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Table 1 Western blot antibody dilution

From: Stimulation of neoplastic mouse lung cell proliferation by alveolar macrophage-derived, insulin-like growth factor-1 can be blocked by inhibiting MEK and PI3K activation

Antibody Target Blocking/Antibody Dilution Buffer* Primary Antibody Dilution† Secondary Antibody Dilution Antibody Source (1°/2°)‡
p-cRaf (S259) TBST + 5% milk 1:2,000 (5% BSA) 1:10,000 goat anti-rabbit CST/CST
c-Raf TBST + 5% milk 1:1,000 (5% BSA) 1:10,000 goat anti-rabbit CST/CST
pErk1/2 (Y204) TBST + 2% milk 1:1,000 1:20,000 goat anti-mouse S.C./S.C.
Erk1/2 TBST + 2% milk 1:50,000 1:40,000 goat anti-rabbit S.C./S.C.
pAkt (S473) TBST + 5% milk 1:4,000 (5% BSA) 1:10,000 goat anti-rabbit CST/CST
Akt TBST + 5% milk 1:10,000 (5% BSA) 1:20,000 goat anti-rabbit CST/CST
pGSK-3β (S9) TBST + 5% milk 1:1,000 (5% BSA) 1:10,000 goat anti-rabbit CST/CST
panGSK-3β TBST + 5% milk 1:2,000 (5% BSA) 1:10,000 goat anti-rabbit CST/CST
Cyclin D1 TBST + 5% milk 1:2,000 (5% BSA) 1:10,000 goat anti-rabbit CST/CST
β-actin TBST + 2% milk 1:100,000 1:100,000 goat anti-mouse Sigma/S.C.
  1. *TBST, 100 mM Tris-buffered saline pH 7.4 with 0.1% Tween-20. † BSA, bovine serum albumin. ‡ Primary (1°) and secondary (2°) antibodies were obtained from Sigma, Santa Cruz Biotechnology, Inc. (S.C.; Santa Cruz, CA) and Cell Signaling Technology (CST).