Anti c myc 9e10

To evaluate the functional activity of hamartin-cmyc expressed by the AAV vector construct (AAV-CMV-hamartin-cmyc), we co-expressed HA-S6 kinase (HA-S6K), which is one of the direct substrates of mTORC1, in 293T cells (Han et al., 2012 (link)). The flag-tagged TSC1 in mammalian expression vector pcDNA3 (TSC1-FLAG) was used as a positive control. Briefly, at 20 h post-transfection, 293T cells were harvested and lysed in 0.5% NP-40 lysis buffer containing 150 mM NaCl, 50 mM Tris (pH 7.4), 50 mM NaF, 1 mM Na orthovanadate, 2 mM EDTA, and 1X Complete protease inhibitor cocktail (Roche). Cell lysates (500 μg) were then subjected to immunoprecipitation (IP) using anti-HA antibody (Covance, Berkeley, CA). Anti-HA immunoprecipitates or cell lysates were separated by 4–15% gradient SDS-PAGE (Bio-Rad, Hercules, CA) and probed with various antibodies, including anti-HA, anti-pS6K (T389) (Cell Signaling, Danvers, MA), anti-C-Myc (9E10, Developmental Studies Hybridoma Bank Iowa), or anti-FLAG (Sigma) antibodies.

Primary antibodies used were the following: anti-c-Myc 9E10 (1:50), anti-Fas2 1D4 (1:50) (Developmental Studies Hybridoma Bank), anti-HA (Covance; 1:500), anti-muscle myosin (1:50), and rabbit anti-β-gal (Cappel; 1:5,000). Alexa Fluor 488 (Molecular Probes), HRP, and Cy3 (Jackson ImmunoResearch Laboratories) conjugated anti-mouse or anti-rabbit secondary antibodies were used at 1:1,000, 1:500, and 1:500, respectively. Cy3-labeled tyramide (PerkinElmer) was used as HRP substrate. ISN projections at embryonic stage 16/17 in A2–A6 abdominal hemisegments were stained with anti-Fas2 and examined in different genetic backgrounds. Stacks of images were obtained with a Zeiss Confocal LSM700 Microscope using a 40× oil immersion objective.