Anti p mek

Parthenolide (PTL), Dehydrocostus, Epoxymicheliolide, Micheliolide, Arglabin, Isoalantolactone and Alantolactone with purity of HPLC ≥ 98% were purchased from Nanjing Spring & Autumn Biological Engineering Co., Ltd (Nanjing, China). Dabrafenib was purchased from MCE Co.,Ltd.(Monmouth Junction, NJ). RPMI 1640, DMEM and Opti-MEM (1×) medium (Gibco, Carlsbad, CA), Block it^™ Alexa Fluor^® Red Fluorescence Control and Lipofectamine^® 2000 (Invitrogen, Carlsbad, CA), TRIzol^® Reagent (Ambion, Carlsbad, CA) were purchased from Thermo Fisher Scientific Inc.. Fetal bovine serum (FBS) were bought from Zhejiang Tianhang Biotechnology Co.,Ltd. (Hangzhou, China). 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) was obtained from MP Biomedicals Inc.. PrimeScript^™ RT Master Mix and SYBR^® Premix Ex Taq^™ (Tli RNaseH Plus) were purchased from Takara Bio Inc. (Dalian, China). The following antibodies were used: anti-Erk 1/2, anti-p-Erk 1/2, anti-MEK, anti-p-MEK, anti-GSK3α/β, anti-GSK3β, anti-c-Myc, anti-Akt, anti-Akt (Ser 473), anti-STAT3, anti-p-STAT3 (Cell Signaling Technology, Inc.), anti-B-Raf (Abcam Inc., Cambridge, UK), anti-GAPDH (Bioworld Technology Inc., St Louis Park, MN). Primers for B-Raf, c-Myc and GAPDH were synthesized by Invitrogen Inc.(Carlsbad, CA).

The indicated cells were incubated with DMSO, 5 µM vemurafenib, 10 µM SHP099, or the combination of 5 µM vemurafenib and 10 µM SHP099 for 0 h, 6 h, 24 h and 48 h. The cells were then lysed in RIPA lysis solution containing protease inhibitors for 30 min, and the protein concentration was determined using BCA reagent. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes and incubated overnight at 4 °C with primary antibodies. The next day, co-incubation with the secondary antibody of the corresponding species was carried out for 2 h. Chemiluminescence detection was performed using the ECL Protein Blotting Assay Kit (Human IgG) (Solarbio). The primary antibodies were used: anti-SHP2 (Cell Signaling Technology, CAT #3397), anti-p-SHP2 (Cell Signaling Technology, CAT #3751), anti-ERK (Cell Signaling Technology, CAT #4695), anti-p-ERK (Cell Signaling Technology, CAT #4370), anti-AKT (Cell Signaling Technology, CAT #4685), anti-p-AKT (Cell Signaling Technology, CAT #4060), anti-MEK (Cell Signaling Technology, CAT #4694), anti-p-MEK (Cell Signaling Technology, CAT #9127).

Caco-2 cells were washed twice with PBS before incubated on ice with 100 μl lysis buffer containing 1% NP40, 50 mM Tris (pH 7.5), 25 mM NaF, 75 mM NaCl, 5 mM MgCl₂, 5 mM EGTA, 0.5 mg/ml aprotinin, 0.1 mM Na₃VO₄, 3 mg/ml Leupeptin, 1 mM DTT. Samples containing 20 μg protein were separated by SDS-PAGE and blotted onto polyvinylidene difluoride membranes using pre-chilled transfer buffer containing 3.6 mg/ml glycine, 7.25 mg/ml Tris base, 0.46 mg/ml SDS, and 20% methanol. The membranes were then immunoblotted, and signals were detected with enhanced chemiluminescence (Thermo Fisher Scientific) and quantified by a LumiImager (Roche Molecular Biochemicals). The anti-p-ERK, anti-p-MEK, anti-total-ERK, anti-total MEK, anti-β-actin antibodies were purchased from CELL Signaling Technology. All antibodies were used at 1:1000 dilution except β-actin, which was used at 1:4000.

Cells were harvested in RIPA buffer containing 100 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS, PH 7.4, and supplemented with protease and phosphatase inhibitor cocktails (Sigma-Aldrich). Protein concentrations were determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein (30 μg) were electrophoresed by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane (Amersham, Piscataway, NJ, USA), and then the membrane was probed with the following antibodies: anti-SS18, anti-GAPDH, anti-HOXC11, anti-Nidogen 2, and anti-β-actin antibodies (Santa Cruz, 1:1000 dilution), anti-SHCBP1 polyclonal antibody (Sigma-Aldrich,1:2000 dilution), anti-p-MEK, anti-MEK, anti-p-ERK, anti-ERK, anti-p-AKT, anti-AKT, and anti-cyclin D1 antibodies (Cell Signaling Technology, 1 : 1000 dilution). Secondary antibodies conjugated to horseradish peroxidase and ECL Western blotting reagents were used for detection.

Total proteins were extracted from cells utilizing RIPA lysis buffer (Cwbio), which contains phenylmethylsulfonyl fluoride (PMSF, Solarbio). Proteins were quantified by the Super-Bardford Pritein Assay Kit (Cwbio). The extractions were loaded into 12% polyacrylamide gel on the Bis-Tris Gel system (Bio-Rad). The products were transferred onto polyvinylidene fluoride (PVDF) membranes, which were then cultivated at 4°C overnight with primary antibodies. The primary antibodies included anti-cleaved-caspase-3 (#ab2303, Abcam, USA), anti-cleaved-PARP (#ab3246, Abcam), anti-MMP-2 (#ab37150, Abcam), anti-vimentin (#ab92547, Abcam), anti-Wnt3a (#ab219412, Abcam), anti-β-catenin (#ab32572, Abcam), anti-t-MEK (#9126, Cell Signaling Technology, USA), anti-p-MEK (#9154, Cell Signaling Technology), anti-t-ERK (#9102, Cell Signaling Technology), anti-p-ERK (#4370, Cell Signaling Technology), and anti-β-actin (#ab179467, Abcam). Then, the PVDF membranes were rinsed and incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (#ab6721, Abcam) and goat anti-mouse IgG (#ab205719, Abcam) for 1 h at 20°C. After washing, the PVDF membranes were treated with ChemiDoc™ XRS system (Bio-Rad), and the intensity of bands was finally evaluated with ImageJ software (NIH, USA).

ARV-771 (S8532), LY3214996 (S8534), SB230580 (S1076), SP600125 (S1460), and sorafenib (S7397) were obtained from Selleckchem (Houston, TX). Anti-CDK4 (ab108357), anti-CDK6 (ab124821), anti-Cyclin D1 (ab16663), anti-p27 (ab32034), anti-Bcl-2 (ab32124), anti-Bcl-XL (ab32370), anti-BRD2 (ab139690), anti-BRD3 (ab50818), anti-BRD4 (ab128874), anti-USP5 (ab154170), anti-USP7 (ab108931), anti-USP8 (ab228572), anti-USP9X (ab19879), anti-USP10 (ab109291), anti-USP13 (ab109624), anti-USP18 (ab168478), anti-USP39 (ab131244), anti-USP14 (ab235960), anti-UCH37 (ab133508), anti-p-ERK (ab32538), anti-ERK (ab184699), anti-p-p38 (ab4822), and anti-p38 (ab170099) were obtained from Abcam (Cambridge, MA). Anti-PARP (#9532), anti-USP1 (#8033), anti-USP15 (#66310), anti-STAMBP (#5245), anti-K48-linked ubiquitin (#12805), anti-ubiquitin (#3936), anti-SIX1 (#16960), anti-SKP2 (#2652), anti-GAPDH (#5174), anti-p-MEK (#3958), anti-MEK (#4694), anti-p-JNK (#9255), and anti-JNK (#9252) were purchased from Cell Signaling Technology (Beverly, MA).