Lumiglo reagent and peroxide

Cells were treated with EGFR-TKI at concentrations of 0.01–1 μmol/L for 4 h. Cells were lysed in Cell Lysis Buffer (Cell Signaling Technology). Equal amounts of protein were loaded per lane on sodium dodecyl sulfate-polyacrylamide gels. Separated proteins were transferred to polyvinylidene fluoride membranes. The membranes were incubated overnight with primary antibodies at 4°C and then incubated with secondary antibodies for 1 h. For the detection of proteins, the membranes were incubated with agitation in LumiGLO reagent and peroxide (Cell Signaling Technology) and then exposed to X-ray film.

Neurons were lysed in sample buffer (1.5 M Tris, 3% SDS, 15% glycerol, 7.5% β-mercaptoethanol, 0.0375% bromophenol blue, pH 6.8) and boiled for 4 min at 100 °C. Gel electrophoresis and western blotting was performed using an Xcell Surelock system with 4–20% NuPage BisTris pre-cast gels (Invitrogen). Post protein migration, the gels were blotted onto polyvinylidene difluoride membranes (Millipore) and then blocked for 1 h at room temperature in 5% (w/v) non-fat dried milk in TBS with 0.1% Tween 20. Membranes were then incubated overnight at 4 °C in blocking solution and diluted primary antibodies: Gclc (1:1000, Abcam), β-actin (1:2000, Abcam). Membranes were then washed in TBS with 0.1% Tween 20, and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Membranes were washed again, and visualized by incubating in LumiGlo reagent and peroxide (Cell Signalling Technology) and exposing Kodak X-Omat film to the membranes.

Cells were lysed in sample buffer containing 60 mM Tris-HCl, pH 6.8, 5% glycerol and 2% SDS. Cell lysates were then boiled for five minutes and protein concentration was measured using a BCA kit purchased from Beyotime (Shanghai, China). Samples were subject to Western blot analysis as previously described [18 (link)]. In brief, equal amount of proteins was loaded and separated on a 7 or 10% SDS-PAGE gel and transferred to a PVDF membrane, which was then blocked with 5% milk for one hour at room temperature. The membrane was incubated overnight at 4°C with primary antibody followed by a secondary horseradish peroxidase-conjugated antibody for one hour at room temperature. Blots were developed using enhanced chemiluminescence (LumiGLO® Reagent and Peroxide, Cell Signaling, Boston, MA, USA) according to the manufacturer’s protocol. Primary antibodies against iNOS, p-JNK1/2, p-p38, p-ERK1/2, p-p65, JNK1/2, p38, ERK1/2, p65, and β-actin, and secondary anti-rabbit or anti-mouse antibody were all purchased from Cell Signaling (Boston, MA, USA).

Cells were treated with EGFR-TKI at concentrations of 0.01–1 μmol/L for 4 h. Cells were lysed in Cell Lysis Buffer (Cell Signaling Technology). Equal amounts of protein were loaded per lane on sodium dodecyl sulfate-polyacrylamide gels. Separated proteins were transferred to polyvinylidene fluoride membranes. The membranes were incubated overnight with primary antibodies at 4°C and then incubated with secondary antibodies for 1 h. For the detection of proteins, the membranes were incubated with agitation in LumiGLO reagent and peroxide (Cell Signaling Technology), and then exposed to X-ray film.

Cell pellets were lysed in 1% Nonidet P40 (IGEPAL), 0.5% sodium deoxycholate, 200 mM NaCl, 10 mM Trizma base, pH 7.8, 1 mM EDTA plus protease inhibitor mixture and 1 mM PMSF for 30 min in ice. Lysates were cleared by centrifugation at 10,000× g for 10 min at 4 °C. Protein concentration was determined by the method of Lowry after precipitation with 5% Trichloroacetic acid (TCA), utilizing bovine serum albumin as a standard. For CFTR analysis 20 μg of total protein was heated in Laemmli buffer (Bio-Rad) at 37 °C for 10 min and loaded onto a 3 to 8% Tris-acetate gel (Bio-Rad). The gel proteins were transferred to PVDF membrane (Bio-Rad) by using Trans Blot Turbo (Bio-Rad) and processed for Western blotting by using mouse monoclonal antibody, clone 596, against NBD2 domain of CFTR (University of North Carolina, Cystic Fibrosis Center, Chapel Hill, NC, USA) at a dilution of 1:2500 by an overnight incubation at 4 °C. After washes, membranes were incubated with horseradish peroxidase-coupled anti-mouse immunoglobulin (R&D System, Minneapolis, MN, USA) at room temperature for 1 h and after washes the signal was developed by enhanced chemiluminescence (LumiGlo Reagent and Peroxide, Cell Signaling). After membranes stripping, β-Actin monoclonal antibody (Sigma-Aldrich) was used to confirm the equal loading of samples [54 (link)].