Phosphorylated erk p erk

The human glioma U-251 cell line was kindly provided by Dr. Dah-Yu Lu (Department of Pharmacology, School of Medicine, China Medical University, Taichung, Taiwan). The human glioma GBM8401 and GBM8901 cell line was kindly provided by Dr. Li-Sung Hsu (Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan). The U-87MG (BCRC No. 60360) and M059K cell line (BCRC No. 60381) were purchased from the Bioresources Collection and Research Center (BCRC, Hsinchu, Taiwan). These cells were cultured in DMEM/F12 and MEM medium supplemented with 1 × penicillin/streptomycin and 10% fetal bovine serum (HyClone, Logan, UT, USA) were incubated at 37 °C in a humidified incubator containing a 5% CO₂ atmosphere. LCN2 antibody was purchased from R&D Systems, Inc (Minneapolis, MN, USA). U0126 (MEK inhibitor), siRNA-CTSD (si-CTSD), Cathepsin D (CTSD), t-ERK, β-actin, HRP-mouse and HRP-rabbit antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Matrigel Matrix was purchased from Corning company (Tewksbury, MA, USA). Phosphorylated-ERK (p-ERK) was obtained from Cell Signaling Technology (Beverly, MA, USA).

Proteins from P19 cells were homogenized in M-PER lysis buffer (Pierce Chemical Co., Rockford, IL). Extracted proteins (15 µg) were separated by SDS-polyacrylamide gel electrophoresis and were electrophoretically transferred onto a membrane according to the previously described approach [20] (link). The membrane was blocked in blocking buffer and incubated with antibodies to NELL2 (Santa Cruz Biotech., Santa Cruz, CA, Catalogue No. sc-54637), β-actin (Sigma-Aldrich, Catalogue No. A5441), Tuj1 (Santa Cruz Biotech., Catalogue No. sc-5274), NeuN (Millipore, Billerica, MA, Catalogue No. MAB377), N-cadherin (Abcam, Boston, MA, Catalogue No. ab76057), phosphorylated ERK (pERK) (CELL Signaling Technology, Beverly, MA, Catalogue No. 9101), ERK (Santa Cruz Biotech., Catalogue No. sc-153), or c-Fos (Santa Cruz Biotech., Catalogue No. sc-7202). Blots were developed using horseradish peroxidase-conjugated anti-goat secondary antibody (Santa Cruz Biotech., Catalogue No. sc-2020), anti-mouse secondary antibody (Santa Cruz Biotech., Catalogue No. sc-2005) or anti-rabbit secondary antibody (Santa Cruz Biotech., Catalogue No. sc-2004). Immunoreactivity was detected with an enhanced chemiluminescence (ECL) kit (Amersham Pharmacia Biotech., Buckinghamshire, UK).

Cells were lysed using cell lysis buffer (Cell Signaling Technology, Beverly, MA, USA). Sample lysates were subject to SDS-PAGE according to a standard protocol. After being transferred to membranes, the samples were immunoblotted with 1000-fold diluted primary antibodies including phosphorylated Akt (p-Akt), Akt (Akt), α-tubulin, phosphorylated Erk (p-Erk), Erk(Erk), phosphorylated p65, α-SMA, TGF-β and β-actin (Cell signaling, MA,USA). The resulting membranes were incubated with secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology, CA, USA). Bands were revealed using an enzyme-linked chemiluminescence detection kit (Amersham Biosciences, Piscataway, NJ, USA) and band density is quantified using an analyser (LumiVision; Aisin, Kariya, Japan).