α c myc

Anti (α)-Akt, α-pAkt, α-Axin, α-β-catenin, α-C3, α-c-Myc, α-cyclin D1, α-GSK3, α-GSK3β, α-pGSK3, α-IL17, α-IL17RC, α-IRS1, α-PI3K, α-pPI3K, α-TAU, α-VEGF, α-Wnt-3a, and α-Wnt-10b were purchased from Santa Cruz. α-c/EBPα, α-pc/EBPα, α-ERK1/2, α-pERK1/2, and α-pIRS1 were bought from Cell Signaling Technology. α-GSK3β was obtained from BD Transduction Lab. GAPDH was purchased from ImmunoChemical. IL17A, IL17F and sFRP2 were bought from R and D. VEGF was obtained from Sigma-Aldrich. IL17A or IL17F (500 ng/mL), sFRP2 (250 ng/mL) or VEGF (10 ng/mL) was applied to cell culture. An IL17RC expression vector was purchased from OriGene.

Protein extracts from infected N. benthamiana leaves were prepared by grinding three discs (0.9 cm diameter) from different leaves in liquid nitrogen and adding 100 μl 4× Laemmli buffer. 20 μl protein samples (2 μl for GFP) were separated on 10% SDS polyacrylamide gels and subjected to immunoblot analyses with α-c-Myc (Santa Cruz Biotechnology Inc., Dallas, USA) or α-GFP (Life Technologies) antibodies. For protein expression analysis of Xcv, bacteria were grown two days on agar plates, then overnight in liquid culture. 500 μl culture (OD₆₀₀ = 0.4) were harvested and the cells resuspended in 50 μl 2× Laemmli buffer. 10 μl protein samples were separated on a 10% SDS polyacrylamide gel and subjected to immunoblot analysis with α-FLAG (Sigma-Aldrich, Taufkirchen, Germany) and α-GroEL (Stressgen, Victoria, Canada) antibodies. α-mouse Ig and α-rabbit Ig were used as secondary antibodies (GE Healthcare Bio-Sciences, Pittsburgh, USA). Antibody reactions were visualized by enhanced chemiluminescence (GE Healthcare).

Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in RIPA buffer (50 mM Tris.HCl, pH 7.4, 150 mM NaCl, 1 % Triton X-100, 1 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate (SDS), 2 mM PMSF, and 1× protease inhibitor cocktail (Roche, Indianapolis, IN)) on ice for 20 min for whole cell lysate preparation. Protein concentration was determined using a Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA), and the optical density at 650 nm was taken using a Bio-Rad iMark microplate reader (Bio-Rad). Cell lysates were electrophoretically separated by 8–12 % sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels and Western blot analysis with the following primary antibodies: α-CREB, α-PCREB, α-BDNF (for the mature form of BDNF), α-p250GAP, α-MecP2, α-synaptophysin, α-PSD-95, α-C-MYC, α-CBP (Santa Cruz Biotechnology, Dallas, TX), α-β-actin (Sigma, St. Louis, MO), α-MAP-2 (EMD Millipore, Billerica, MA), α-His (Applied Biological Materials ABM, Richmond, BC), and α-p24 (NIH AIDS Reagents Program, donated by Dr. Bruce Chesebro of NIAID) [73 (link)]. Sheep anti-mouse IgG-HRP and donkey anti-rabbit IgG-HRP from GE Healthcare (Marlborough, MA) were used as secondary antibodies, followed by ECL detection and imaging using a Bio-Rad ChemiDoc imaging system (Bio-Rad).