Western bright ecl detection system

Cells were treated with trametinib at the indicated concentrations and times, and then lysed in RIPA buffer containing protease inhibitors. Equal quantities (150 μg protein per lane) of total proteins were subjected to 10% SDS-PAGE, and then electrophoretically transferred onto PVDF membranes (Roche Diagnostics, Mannheim, Germany). The membranes were then incubated with the indicated primary antibodies at 4°C overnight. Anti-p-ERK1/2 and anti-total-ERK1 (t-ERK) were purchased from Bioworld Technology. Anti-GAPDH was purchased from Abmart. Anti-β-catenin was purchased from Cell Signaling Technology. This was followed by incubation with species-specific HRP-conjugated secondary antibodies from ZSGB-BIO, and antigen-antibody complexes were visualized using the Western Bright ECL detection system (Advansta, CA).

Cells were lysed in prechilled RIPA buffer containing protease inhibitors, and equal amounts of protein lysates were separated by 10% SDS-PAGE and transferred to PVDF membranes (Roche Diagnostics, Mannheim, Germany). Next, we incubated the membranes with the indicated primary antibodies (Supplementary Table S3) at 4 °C overnight. After incubation the membranes with species-specific HRP-conjugated secondary antibodies (ZSGB-BIO, Beijing, China), we used the Western Bright ECL detection system (Advansta, CA) to visualize the immunoblotting signals.

Total proteins from murine samples were obtained using TriPure Reagent (Roche Applied Science), following manufacturer's instructions. Total proteins from cell lines were obtained using radioimmunoprecipitation assay (RIPA) cell lysis buffer. Protein extracts were supplemented with 2 mM phenylmethylsulphonyl fluoride (PMSF), 2.5 μl/ml Protease Inhibitor Cocktail and 10 μl/ml Phosphatase Inhibitor Cocktail 2 (Roche Diagnostics GmbH, Mannheim, Germany). Ten micrograms-aliquots were electrophoresed in 12% SDS-PAGE with β-mercaptoethanol, then electrotransferred to Immobilon-P transfer membranes (Merck Millipore).
The peroxidase activity was developed using WesternBright ECL Detection System (Advansta, Menlo Park, CA, USA). ImageQuant LAS 4000 digital imaging system (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) was used for acquisition of images, and Scion Image Software (Scion Corporation, NIH, Frederick, MD, USA) for band densitometry.

Cells were lysed in prechilled RIPA buffer containing protease inhibitors. Equal amounts of protein lysates were separated by SDS–PAGE and transferred onto PVDF membranes (Roche Diagnostics, Mannheim, Germany). The membranes were then incubated with primary antibodies. Anti-AIB1 and anti-total-Erk1/2 (t-Erk) were purchased from Abcam, Inc. Anti-phospho-Akt^Ser473, anti-phospho-Erk1/2 and anti-total-Akt (t-Akt) were purchased from Bioworld Technology, co, Ltd. Anti-total-Rb (t-Rb) and anti-phospho-Rb^S811 (p-Rb) were purchased from Epitomics, Inc. Anti-GAPDH was purchased from Abgent, Inc. This was followed by incubation with species-specific HRP-conjugated secondary antibodies from ZSGB-BIO, and immunoblotting signals were visualized using the Western Bright ECL detection system (Advansta, CA).

Proteins were electrophoresed in Mini-PROTEAN-TGXTM Precast Gels (Bio-Rad Laboratories, Hercules, CA, USA) and then transferred to mini-sized PVDF membranes by the Tranfer Blot^® Turbo^TM Transfer System (Bio-Rad Laboratories). The peroxidase activity was developed using the WesternBright ECL Detection System (Advansta, Menlo Park, CA, USA). The ImageQuant LAS 4000 digital imaging system (GE Healthcare Europe GmbH, Freiburg, Germany) was used for acquisition of images and Quantity One^® v4.6.3 (Bio-Rad Laboratories) for band densitometry. The primary antibodies used for immunodetection were the anti-FADD antibody (FADD (1F7): 05-486; Merck Millipore, Billerica, MA, USA) and anti-DIABLO (Smac (C-10): sc-393118; Santa Cruz Biotechnology, Dallas, TX, USA). The secondary antibody was anti-mouse IgG-HRP (#7074; Cell Signaling Technology, Danvers, MA, USA).

Treated cells were lysed in prechilled RIPA buffer containing protease inhibitors.
Equal amount of proteins was electrophoresed using 10 % SDS-PAGE, and transferred onto polyvinylidene fluoride (PVDF) membranes (Roche Diagnostics, Mannheim, Germany). The membranes were blocked in 5 % BSA dissolved in TBST (1 × TBS buffer and 0.05 % Tween 20) for 2 h, and then incubated at 4 ℃ overnight with the indicated primary antibodies. Anti-phospho-Akt (Ser473 or Thr308; p-Akt) and anti-total Akt (t-Akt) antibodies were purchased from Bioworld Technology (St. Louis, MO, USA). Anti-total mTOR (t-mTOR), anti-p53, anti-β-catenin, anti-phospho-β-catenin (Ser33; p-β-catenin) and anti-Cdk2 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-phospho-mTOR (Ser2448; p-mTOR), anti-FOXO3a, anti-phospho-FOXO3a (Thr32; p-FOXO3a), anti-p21, anti-GSK3β and anti-phospho-GSK3β (Ser9; p-GSK3β) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-cyclin E1 antibodies was purchased from Sino Biological (Beijing, China). Anti-GAPDH was purchased from Abmart (Shanghai, China). Then species-specific HRP-conjugated secondary antibodies were incubated (ZSGB Biotechnology, Beijing, China) and immunoblotting signals were visualized using the Western Bright ECL detection system (Advansta, CA, USA).

Cells were lysed in prechilled RIPA buffer containing protease inhibitors. The protein lysates were separated on SDS–PAGE and then transferred to PVDF membranes (Roche Diagnostics, Mannheim, Germany). The membranes were blocked for 2 h in 5% bovine serum albumin (BSA) in 1 × TBS-T (0.5% Tween-20) and incubated with the indicated primary antibodies, including anti-EHF (Abcam, Inc), anti-total-Erk1/2 (Abcam, Inc), anti-phospho-Erk1/2 (Epitomics, Inc), anti-phospho-AktSer473 (Bioworld Technology, co, Ltd), anti-total-Akt (Bioworld Technology, co, Ltd), anti-HER2 (Sino Biological, Inc), anti-HER3 (Sino Biological, Inc), anti-HER4 (Sino Biological, Inc), anti-E-cadherin (Epitomics, Inc), anti-Vimentin (Epitomics, Inc) and anti-GAPDH (Abgent, Inc). The membranes were then incubated with species-specific HRP-conjugated secondary antibodies from ZSGB-BIO, and immunoblotting signals were visualized using the Western Bright ECL detection system (Advansta, CA).