Protease inhibitor and phosphatase inhibitor

BM cells were lysed in a RIPA Buffer (Biosesang, Seongnam, Republic of Korea) including a protease inhibitor and phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA), and they were sonicated on ice. After centrifugation, clear cell lysates were acquired and assayed for protein concentration. Following denaturation, 40 µg of protein was separated on a 4–12% SDS-PAGE gel (Invitrogen, Waltham, MA, USA) at 150 V for 90 min and transferred to a methanol-soaked PVDF membrane (Invitrogen) at 40 V for 100 min. The membranes were blocked with 3% bovine serum albumin for 1 h and then incubated at 4 °C overnight with anti-phopho-SAPK/JNK (Thr183/Tyr185) (#9255, Cell Signaling Technology, Danvers, MA, USA), anti-phopho-p38 MAPK (Thr180/Tyr185) (#9216, Cell Signaling Technology), anti-SAPK/JNK (#9252, Cell Signaling Technology) and anti-p38 MAPK (#9212, Cell Signaling Technology) Abs. The membranes were then incubated with anti-mouse or anti-rabbit IgG Abs conjugated to horseradish peroxidase (HRP) at room temperature for 1 h. After stripping at 55 °C for 30 min, they were incubated at room temperature for 1 h with anti-β-actin Abs (Santa Cruz Biotechnology, Dallas, TX, USA) and subsequently with anti-mouse HRP Abs (Cell Signaling Technology). The protein expression levels were normalized to the corresponding β-actin levels.

After removing the medium, cells were washed thoroughly with ice-cold PBS. Then the cells were harvested in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM sodium chloride, 1% NP40, 1 mM EDTA, 5 mM sodium fluoride, 0.25% sodium deoxycholate, 5 mM sodium orthovanadate, 0.4% SDS, 1 mM PMSF) containing protease inhibitor and phosphatase inhibitor (Thermo, USA) for 2 h at 4°C. The concentration of proteins was determined by the Bradford assay then cell lysates were subjected to 10% or 12% SDS-PAGE and transferred to nitrocellulose membranes (Whatman International, Ltd.). The membrane was blocked with 5% non-fat milk powder in TBST buffer (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) for 1h at room temperature then was incubated with primary antibodies at 4°C overnight. The membrane was incubated with HRP-conjugated secondary antibodies for 1 h at room temperature, the antibody-antigen complex on the membrane was visualized by enhanced chemiluminescence system(Thermo).

Whole‐cell lysates were prepared in M‐PER buffer supplemented with protease inhibitor and phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA), resolved by SDS/PAGE, and blotted with indicated antibodies. SuperSignal West Pico Chemiluminescent Substrate and SuperSignal Western Blot Enhancer (Thermo Fisher Scientific) were used to enhance blotting signal when needed. The following antibodies were used in this study: anti‐MAP4K4, anti‐phospho‐ERK1/2, anti‐phospho‐JNK, anti‐JNK, anti‐phospho‐p38, anti‐p38, anti‐AKT, anti‐phospho‐AKT, anti‐MEK1/2, anti‐phospho‐MEK1/2, anti‐RAS and anti‐PP2A‐C (Cell Signaling Technology, Danvers, MA, USA); anti‐ERK1/2, anti‐GAPDH, anti‐β‐actin, anti‐PP2A‐B56β, and anti‐MKP3 (Santa Cruz Biotechnology, Dallas, TX, USA); anti‐6Χ HIS (Immunology Consultants Laboratory, Inc., Lake Oswego, OR, USA). Erlotinib and trametinib were purchased from Selleck Chemicals (Houston, TX, USA). The protein phosphatase 2 (PP2A) inhibitor okadaic acid (OA) was purchased from Sigma Aldrich. The PP2A activator FTY720 was purchased from Cayman Chemical (Ann Arbor, MI, USA). MAP4K4 inhibitor PF‐06260933 was purchased from Tocris Bioscience (Avonmouth, Bristol, UK). EGF was obtained from Thermo Fisher Scientific.

Hippocampal was homogenized in RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCL, PH 8.0) containing protease inhibitor and phosphatase inhibitor (Thermo). Lysates were then dissolved in 2 X laemmli sample buffer (Biorad), and boiled at 95 °C for 5 minutes. For western blot, lysates (50 ug protein) were added in SDS-PAGE and electroblotted onto PVDF membrane. The membranes were blocked with 5% skim milk in TBS-T, and then probed with desired antibodies. Primary antibodies against p-p38 (Cell Signaling), p38(Anbo) and β-actin (Sigma) were used. Immobilon Western Chemiluminescent HRP Substrate (Merck) was used to reveal the antibody-antigen complexes.