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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).
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Molecular Cancer

ISSN: 1476-4598