Rabbit anti mouse igg

For determination of isotypes, wells of microtitration plate (Lynbro/Titertek) were coated with N. meningitidis strain L7 LOS in 0.05 M carbonate buffer pH 9.6 (2 μg/100 μl/well) at 37 °C for 3 h and then at 4 °C overnight. The plate was then blocked with 1% BSA in T-TBS (250 μl/well) at room temperature for 1 h. After threefold washing with T-TBS (250 μl/well), the plate was incubated for 2.5 h at room temperature with murine antisera (100 μl/well) diluted 1:100 with PBS. The plate was then washed five times with T-TBS (250 μl/well), followed by incubating for 1 h at room temperature with rabbit anti-mouse IgG (Bio-Rad, USA) subclass (IgG1, IgG2a, IgG2b, IgG3, IgM, IgA) specific antiserum (100 μl/well). After washing with T-TBS, 100 μl of 1:3000 dilution with PBS of a horseradish peroxidase-labeled goat anti-rabbit IgG (H + L chains, Kirkegaard & Perry Laboratories, USA) was added to each well. Following the incubation for 1 h at room temperature the plate was washed with T-TBS and TMB peroxidase substrate (Kirkegaard & Perry Laboratories) was added (100 μl/well). The reaction was developed at room temperature and stopped with 1 M H₃PO₄ (50 μl/well). Optical densities were measured at 450 nm using Dynatech MR 5000 microplate reader.

The human NSCLC H1299, H358, H1975 and Calu-3 cell lines were provided by American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in RPMI 1640 (Sigma-Aldrich) medium supplemented with 10% fetal bovine serum (FBS; Life Technologies, Gaithersburg, MD) in a humidified atmosphere with 5% CO2. The identity of all cell lines was confirmed by STR profiling (Promega) on an ad hoc basis prior to performing experiments, and repeated after the majority of the experiments were performed.
Metformin was purchased from Sigma-Aldrich; Selumetinib (AZD6244) and Pimasertib (AS-703026) from Selleck Chemicals (Selleckchem, Houston, TX, USA). They were dissolved in sterile dimethylsulfoxide (DMSO) and a 10 mM stock solution was prepared and stored in aliquots at −20°C. Working concentrations were diluted in culture medium just before each experiment.
Recombinant Sonic Hedgehog was provided by Sigma-Aldrich.
Primary antibodies for western blot analysis against p-EGFR (Tyr1068), EGFR, p-MEK1/2 (Ser217/221), MEK1/2, p-MAPK44/42 (Thr202/Tyr204), MAPK44/42, p-AKT (Ser473), AKT, AMPK, p-AMPK (thr172), S6, p-S6 (Ser235-236), Vimentin, Snail, GLI1 and β-Actin were obtained from Cell Signaling Technology. The following secondary antibodies from Bio-Rad were used: goat anti-rabbit IgG and rabbit anti-mouse IgG.

Protein lysates containing equal amount of proteins, measured by a modified Bradford assay (BIORAD, Hercules, CA), were subjected to direct Western Blot (WB). Immuno-complexes were dectected with the enhanced chemiluminescence kit (ECL plus, Thermo Fisher Scientific, Rockford, IL). We used the following antibodies from Cell Signalling (Beverly, MA): anti-EGFR, anti-phospho-EGFR (Tyr1068), anti-HER2, anti-phospho-HER2 (Tyr1248), anti-HER3, anti-phospho-HER3 (Tyr1289), anti-IGF1R-beta, anti-phospho-IGF1R-beta (Tyr1135), anti-p44/42 MAPK, anti-phospho-p44/42MAPK, anti-AKT, anti-phospho-AKT (Ser 473), anti-AXL, anti-c-MET, anti-S6 ribosomal protein, anti-phospho-S6 ribosomal protein, anti-4EBP1, anti-phospho-4EBP1, anti-vimentin, anti-E-cadherin, anti-Snail. Anti-α-tubulin (internal loading control) was from Sigma (Sigma-Aldrich, St. Louis, MO). The following secondary antibodies from Biorad were used: goat anti-rabbit IgG and rabbit anti-mouse IgG. Each experiment was done in triplicate.