Goat anti mouse rabbit irdye

Cell lysate was prepared in RIPA buffer (50 mM Tris-HCl pH8.0, 150 mM NaCl, 0.1% SDS, and 0.5% Na deoxycholate, 1% NP40) with fresh proteinase inhibitor cocktail from Roche (Basel, Switzerland). Samples were quantified by Bradford reagent from Sigma (St. Louis, MO), and measured at 595 nm with a microplate reader. Equal amount of protein (10 μg) was loaded. After standard transfer and block, the sample membrane was incubated with primary antibody for overnight at 4°C. After standard wash, the membrane was incubated with secondary antibody Goat anti-mouse/Rabbit IRDye from Li-Cor. IRDye fluorescent dyes have absorption and emission wavelengths in the near-infrared spectrum, between 680 and 800 nm. The western signals can be detected and analyzed using a Li-Cor Odyssey image reader by software Image Studio (Ver. 2.1) from Li-Cor (Lincoln, NE). Antibodies used: LC3A/B (4108, dilution 1:1000), Atg3 (3415, dilution 1:1000), and GRP78 (3177, dilution 1:1000) from Cell Signaling (Danvers, MA), β-Actin (AC-15, dilution 1:3000) from Santa Cruz (Dallas, TX), Goat anti-mouse/Rabbit IRDye (dilution 1:10000) from Li-Cor (Lincoln, NE).

Cell lysate was prepared in RIPA buffer (50 mM Tris-HCl pH8.0, 150 mM NaCl, 0.1% SDS, and 0.5% Na deoxycholate, 1% NP40) with fresh proteinase inhibitor. The membrane fraction was extracted using Mem-PER Plus Membrane Protein Extraction Kit from ThermoFisher Scientific (Waltham, MA) according to the manufacturer’s manual. The nuclear and cytoplasmic fractions were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents from ThermoFisher Scientific (Waltham, MA) according to the manufacturer’s manual. Samples were quantified by Bradford reagent from Sigma (St. Louis, MO) and measured at 595 nm with a microplate reader. Equal amount of protein was loaded. Western detection was carried out using a Li-Cor Odyssey image reader by software Image Studio (Ver. 2.1) from Li-Cor (Lincoln, NE). Antibodies used: β-catenin (D10A8, dilution 1:1000), E-Cadherin (24E10, dilution 1:1000), Histone H3 (D1H2, dilution 1:2000), Cyclin D1(2922, dilution 1:1000), Na,K-ATPase (3010, dilution 1:1000), Phospho-Rb (9307, dilution 1:1000) from Cell Signaling (Danvers, MA), Myc (9E10, dilution 1:1000), β-Actin (AC-15, dilution 1:3000) from Santa Cruz (Dallas, TX), Goat anti-mouse/Rabbit IRDye (dilution 1:10000) from Li-Cor (Lincoln, NE).

Cell lysate was prepared in RIPA buffer (50 mM Tris-HCl pH8.0, 150 mM NaCl, 0.1% SDS, and 0.5% Na deoxycholate, 1% NP40) with fresh proteinase inhibitor. Equal amount of protein was loaded. Western detection was carried out using a Li-Cor Odyssey image reader by software Image Studio (Ver. 2.1) (Lincoln, NE). Antibodies used: phospho-S6K (Thr389) (dilution 1:1000) from Millipore (Billerica, MA), S6K (H-9,dilution 1:2000), TSC2 (C-20, dilution 1:1000), β-Actin (AC-15, dilution 1:3000) from Santa Cruz (Dallas, TX), Rb (4.1, dilution 1:10) from Hybridoma Bank (Iowa City, IA), Goat anti-mouse/Rabbit IRDye (dilution 1:10000) from Li-Cor (Lincoln, NE).