Horseradish peroxidase conjugated rabbit igg

Cells from HEK293, HeLa, H9C2 and neonatal cardiac myocytes were lysed in extraction buffer (0.2% Triton X-100, 50mM NaCl, 5mM EDTA, 50mM Tris at pH 7.4 and complete EDTA-free protease inhibitor cocktail (Roche). Extracts were mixed with SDS sample buffer for degradation. The proteins were resolved by SDS-PAGE and transferred to nitrocellulose membrane and blotted for anti-β1AR (ab-3442- 1:1000), anti-SLC22A3 (ab124826-1:1000) or GAPDH (1:10,000) antibodies to detect β1AR, EMT/OCT3 and GAPDH expression by horseradish-peroxidase-conjugated rabbit IgG, sheep anti-mouse and rabbit IgG (1:10,000 Amersham Biosciences) and SuperSignal extended duration detection reagent (Pierce).

Human myeloma IgE (huIgE, Calbiochem, La Jolla, CA) was biotinylated in the NIAID Core Facility. The protein-specific antibodies used in confocal microscopy were: mTOR (10343, IBL, Minneapolis, MN) and rictor (NB100-612, Novus Biologicals, Littleton, CO). The β-actin-specific antibody for immunoblotting was from Cell Signaling Technology (Beverley, MA) and Lyn-specific antibody for immunoblotting from Santa Cruz Biotechnology (Santa Cruz, CA). The conjugated protein-specific antibodies were: fluorophore-conjugated antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), horseradish peroxidase-conjugated rabbit IgG (Amersham Biosciences, Piscataway, NJ), and mouse IgG Fc-specific antibody (Sigma-Aldrich). The phosphoprotein-specific antibodies for immunoblotting were: p-mTOR(Ser2448), p-mTOR(Ser2481), p-rictor (Thr1135), p-Akt(Thr308), p-Akt(Ser473), p-LAT(Tyr171), p-S6 ribosomal protein (S6RP) (Ser240/244), p-4E-BP1(Thr37/46), and rictor (Cell Signaling Technology), and p-PLCγ₁(Tyr783) (Invitrogen). The blocking donkey serum was from Santa Cruz Biotechnology and Torin1 (37 (link)) was a gift of Dr. Nathanael S. Gray, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA. FITC-conjugated phalloidin and other chemicals and reagents were from Sigma-Aldrich.