Phospho erk

Total RNA was isolated using Trizol (Invitrogen), according to the manufacturer’s protocol, and the nuclear and cytoplasmic fractions were prepared according to the manufacturer’s instructions (Promega Corporation). Real-time reverse-transcription PCR assay was performed with SYBR Green PCR Master Mix using a StepOnePlus Detection System (ABI) to detect MALAT1, KRAS, miR-217 and GAPDH mRNA expression levels. All the primers used in this study are listed in Supplementary Table S2. Western blotting analysis was performed as described previously^{52 (link)}. Primary antibodies against the following proteins were used in this study: KRAS, RAS, phospho-MEK, phospho-ERK and β-actin (the antibodies against KRAS and β-actin were purchased from Santa Cruz Biotechnology Inc., and the other antibodies were purchased from Cell Signaling Technology).

Curcuma Longa Linn (powdered form) and lecithin (L-α-phosphatidylcholine) were obtained from Sigma-Aldrich (St. Louis, MO, USA). The organic solvents such as toluene and dichloromethane were purchased from Fisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) and phosphate-buffered saline (PBS) were purchased from GE Healthcare (Logan, UT, USA). The following antibodies were obtained: c-Src, phospho-c-Src, PKC, phospho-PKC, JNK, phospho-JNK, p38, phospho-p38, ERK, phospho-ERK, IκBα, phospho-IκBα, NF-κBp65, phospho-NF-κBp65, Bcl-2, Bax, cleaved caspase-3, and β-actin antibodies (Santa Cruz Biotechnology, Paso Robles, CA, USA); The following reagents were obtained: N-acetylcysteine (NAC) (Tocris, KOMA Biotech, Seoul, Korea) and 5-(and-6)-chloromethyl-2′,7′- dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA) (Invitrogen, Carlsbad, CA, USA). PP2, SP600125, Bisindolylmaleimide I, and Bay11-7082 were obtained from MedChemExpress (Monmouth Junction, NJ, USA). All other reagents did not show any critical cytotoxic effects by themselves.

Here is the information regarding the chemicals and antibodies utilized in the procedure: Amyloid-β (Santa Cruz, Dallas, TX, United States, sc-53822, 1:200), BACE1 (Invitrogen, Waltham, MA, United States, PA5-19952, 10 μg/mL), APP (Invitrogen, 14–9,749-82, 2.5 μg/mL), BACE1 (Invitrogen, PA5-19952, 1:1000), Iba-1 (abcam Cambridge, CAM, UK, ab178846, 1:2000), GFAP (BD Biosciences, Dickinson, ND, United States, BD-556328, 5 μg/mL), β-actin (Santa Cruz, sc-8432, 1:1000), phospho-JNK (Santa Cruz, sc-6254, 1:1000), JNK (Cell signaling, cs-9258, 1:1000) phospho-ERK (Santa Cruz, sc-7383, 1:1000), ERK (Cell signaling, cs-9102, 1:1000) phospho-p38 (Cell Signaling, Danvers, MA, United States, csD3F9, 1:1000), p38 (Santa Cruz, sc-535, 1:1000) Anti-goat mouse (Enzo Life Sciences, Farmingdale, NY, United States, ADI-SAB-100-J, 1:2000), Anti-goat rabbit (Enzo Life Sciences, ADI-SAB-300-J, 1:2000).

NPs and cellular protein total protein concentration evaluated by Pierce^TM BCA Protein Assay Kit (Thermo Fisher). NP and cell lysates were loaded into each well and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 10%). The protein bands were transferred to nitrocellulose membranes after which were blocked in 5% skim milk or 5% BSA. Primary antibodies (dilution 1:1000) against CD 63 (Abcam, Cambridge, UK), HSP 70, phospho-AKT, AKT, phospho-ERK, ERK (Santa Cruz, Dallas, TX, USA), or β-Actin (Abcam, Cambridge, UK) were incubated with the membrane at 4 °C overnight. After being washed three times with TBST (TBS with 0.1% Tween20^®), membranes were incubated with horseradish peroxidase-linked anti-mouse or anti-rabbit secondary antibody for 1 h at room temperature. After being washed three times with TBST for 15 min each, the reactivity was examined by an enhanced chemiluminescence kit (Thermo Fisher Scientific, Waltham, MA, USA). The image of the membrane was taken using UV or white light on a Davinci-K Gel Imaging System (Davinch-K, Seoul, Korea). The density of bands was quantified by Image J (Version 1.50, National Institutes of Health, Bethesda, MD, USA).

Lapatinib was purchased from LC Laboratories (Woburn, MA). The following antibodies were purchased from Cell Signaling (Danvers, MA): EGFR, phospho-EGFR (Ser1046/1047), phospho-erbB-2 (Tyr877), phospho-erbB-3 (Tyr1289), phospho-Akt (Ser473), and phospho-ERα (Ser167); Millipore (Temecula, CA): ERα and erbB-2; and Santa Cruz Biotechnology (Santa Cruz, CA): erbB-3, c-Myc, Erk2, phospho-Erk, Bcl-2, cyclin D1, and β-actin. Horseradish peroxidase (HRP)-labeled goat anti-rabbit and anti-mouse secondary antibodies were used from Thermo Scientific (Rockford, IL).