Actin hrp sc 47778

Cells were lysed directly in-well using RIPA buffer (10 mM Tris-HCl, 150 mM NaCl, 1% Triton, 1% sodium deoxycholate, 0.1% SDS, pH 7.2) supplemented with 10 μg/mL aprotinin, 5 μg/mL leupeptin, 7 μg/mL pepstatin, 10 mM NaF, 2 mM sodium orthovanadate, 10 mM β-glycerophosphate, and 2 mM sodium pyrophosphate). Lysates were then sonicated thoroughly on ice. Protein concentrations were normalized by the BCA assay (Pierce), resolved on 4–20% Tris-glycine gels (Invitrogen), transferred to 0.45-μm PVDF (Thermo) using Towbin’s buffer (low amperage for ~4 h at 4 °C), blocked with 5% non-fat milk in TBST, then probed with primary antibodies overnight at 4 °C. Antibodies used in this study were the following: Actin-HRP (sc-47778) from Santa Cruz Biotechnology; CDK4 (12790), Cyclin D2, (3741), 4E-BP1 (9644), p4E-BP1 (T37/46) (2855), p4E-BP1 (S65/101) (9451), p4E-BP1 (T70) (9455), Rb (9313), pRb (S780) (3590), eIF4E (9742), eIF4G (2498), and pS6 (240/244) (2215) from Cell Signaling Technology.

MCF-7, MDA-MB-231, MDA-MB-468 cells were lysed directly in-well using RIPA buffer (10 mM Tris-HCl, 150 mM NaCl, 1% Triton, 1% sodium deoxycholate, 0.1% SDS, pH 7.2) supplemented with 10 μg/mL aprotinin, 5 μg/mL leupeptin, 7 μg/mL pepstatin, 10 mM NaF, 2 mM sodium orthovanadate, 10 mM β-glycerophosphate, and 2 mM sodium pyrophosphate). Lysates were then sonicated thoroughly on ice. Protein concentrations were normalized by the BCA assay (Pierce), resolved on 4–20% Tris-glycine gels (Invitrogen), transferred to 0.45-μm PVDF (Thermo) using Towbin’s buffer (low amperage for ~4 h at 4 °C), blocked with 5% non-fat milk in TBST, then probed with primary antibodies overnight at 4 °C. Antibodies used in this study were the following: Actin-HRP (sc-47778) and Myc-9E10 (sc-40) from Santa Cruz Biotechnology; CDK4 (12790), CDK6 (13331), Cyclin D2, (3741), DYKDDDK tag (14793), eIF4E (9742), eIF4G (2498), MEK1/2 (9122), mTOR (2972), S6 (2217), pS6 (240/244) (2215), p4E-BP1 (T37/46) (2855), p4E-BP1 (S65/101) (9451), p4E-BP1 (T70) (9455), SAPK/Jnk (9252), 4E-BP1 (9644), c-Myc (13987), Rb (9313) and pRb (S780) (3590) from Cell Signaling Technology; and FLAG-M2 (F1804) from Sigma. All experiments were performed in biological triplicate.