Rabbit anti phospho p70 s6 kinase thr389

Mouse monoclonal anti-Flag M5 antibody was from Sigma-Aldrich. Rabbit anti-phospho-Smad2 and mouse anti-Myc were home-made and have been described before [59 (link)]. Mouse anti-HA was from Roche. Rabbit anti-HA and mouse anti-LKB1 used for co-immunoprecipitation and IHC assays, mouse anti-Smad1, rabbit anti-TGFβRI/ALK5 (V-22), rabbit anti-ID1, goat anti-STRADα, goat anti-Smad7, mouse anti-β-tubulin, mouse anti-ubiquitin (P4D1), and rabbit and mouse IgGs used for control immunoprecipitations, were from Santa Cruz Biotechnology. Mouse anti-BMPRIA/ALK3, mouse anti-BMPRIB/ALK6 and goat IgG used for control immunoprecipitations and immunoblotting, were from R&D Systems, Inc. Mouse monoclonal anti-GAPDH was from Ambion. Mouse anti-E-cadherin was from Becton Dickinson Transduction Labs. Rabbit anti-phospho-Smad1/5/8, rabbit anti-phospho-AMPK (Thr172), rabbit anti-AMPKα, rabbit anti-phospho-p70 S6 kinase (Thr389), rabbit anti-p70 S6 kinase and rabbit anti-ACVRI/ALK2 were from Cell Signaling Technology, while rabbit anti-MO25 and anti-Smad1 were from Epitomics, and were used for immunoprecipitation, immunoblot and IHC assays.

Snap frozen embryos or brains for protein extraction were minced in extraction buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1%SDS, 50 mM Tris-HCl pH 7.5) and incubated at 4 °C for 30 minutes. Lysates were separated on Invitrogen Bolt precast 4–12% polyacrylamide gels and transferred to PVDF membrane before blotting. Antibodies and their corresponding dilutions were: rabbit anti phospho-S6 Ribosomal Protein (Ser235/236) (Cell Signalling, #2211, 1:1000) rabbit anti phospho-S6 Ribosomal Protein (Ser240/244) (Cell Signalling, #5364, 1:1000), rabbit anti phospho-p70 S6 Kinase (Thr389) (Cell Signalling, #9234, 1:1000), rabbit anti phospho-AKT (Ser473) (Cell Signalling, #4060, 1:2000), rabbit anti phospho-AKT (Thr308) (Cell Signalling, #13038, 1:750), rabbit anti p70 S6 Kinase (Cell Signalling, #9202, 1:500), rabbit anti S6 Ribosomal Protein (Cell Signalling, #2217, 1:750) and rabbit anti mTOR (Cell Signalling, #2983, 1:1000).

For Western blotting and immunofluorescence, the following primary antibodies were used: rabbit anti-LC3 [PM036] (1:100 for immunofluorescence; 1:500 for Western blot), rabbit anti-phospho-p62 [PM074] (Ser351) (1:500) from Medical & Biological Laboratories (MBL); rabbit anti-mTOR [#2972] (1:1000), rabbit anti-phospho-mTOR (Ser2448) [#2971] (1:100 for immunofluorescence; 1:1000 for Western blot), rabbit anti-p70S6 kinase [#2708] (1:1000), and rabbit anti-phospho-p70S6 kinase (Thr389) [#2708] (1:1000) from Cell Signaling; guinea pig anti-p62 [P0G-GP62-C] (1:1000) from Progen; rabbit anti-ATP6V0A4 [ab97440] (1:1000) from AbCam; mouse anti-β-tubulin [T4029] (1:1000) and mouse anti-β-actin [A5316] (1:1000) from SIGMA; and rabbit Spin1/SPNS1 (1:100) from original antibodies^{12 (link)}.