Peroxidase coupled secondary antibodies

Whole cell lysates were prepared from E10.5 embryo head or E9.5 embryo using NP40 lysis buffer (50 mM Tris-HCl, pH7.4, 200 mM NaCl, 2 mM MgCl₂, 1% Nonidet P-40 (NP-40), 1 mM PMSF, 1 μg/ml leupeptin, 2 μg/ml aprotinin, and 1 μg/ml pepstatin). Proteins were separated on Novex 4–20 % Tris-Glycine Gel (Invitrogen) and transferred to PVDF membrane. The membranes were incubated with 5% milk for 1 h and incubated with primary antibodies overnight at 4°C. Primary antibodies were used as follows: phospho-Smad1/5/9 antibody (1:1000, #13820, Cell Signaling), phospho-mTOR (Ser2448) (1:1000, #2971, Cell Signaling), mTOR (1:1000, #2972, Cell Signaling), phospho-S6K1 (Thr389) (1:500, #9205, Cell Signaling), S6K1 (1:1000, #9202, Cell Signaling), phospho-S6 (Ser235/236) (1:2000, #2211, Cell Signaling), phospho-S6 (Ser240/244) (1:2000, #5364, Cell Signaling), S6 (1:2000, #2217, Cell Signaling), TSC1 (1:1000, #6935, Cell Signaling), and β-actin (1:1000, #4970, Cell Signaling). Blots were incubated with peroxidase-coupled secondary antibodies (Promega) for 1 h, and protein levels were detected with SuperSignal West Pico or Femto Chemiluminescent Substrate (Thermo Scientific). Images were quantified by ImageJ software (NIH).

Whole cell lysates were prepared from cells, separated on SDS-polyacrylamide gel and transferred to PVDF membranes. The membranes were incubated with 5% milk in PBS for 1 hr and then incubated with primary antibodies overnight at 4°C. Blots were incubated with peroxidase-coupled secondary antibodies (Promega) for 1 hr, and protein expression was detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).