Anti tigar

Protein samples were isolated from colorectal cancer tissues using PARIS kit (Ambion). Total protein concentration was determined using Bradford protein reagent (Bio-Rad). Soluble proteins were loaded on precast TGX gels and were analyzed by immunoblotting with anti-TIGAR (dilution 1:1,000; Santa Cruz Biotechnology). Reactivity was detected with horseradish peroxidase-conjugated secondary antibodies and chemiluminescence (GE Healthcare). Membrane was developed using C-Digit Blot Scanner (LI-COR, Hamburg, Germany). CRC cell line whole cell lysates were prepared as described (22 (link)). Soluble proteins were analyzed by immunoblotting with anti-TIGAR (Santa Cruz Biotechnology) and anti-β-actin (Sigma). Reactivity was detected with horseradish peroxidase-conjugated secondary antibodies and chemiluminescence (GE Healthcare). Membranes were developed using C-Digit Blot Scanner (LI-COR).

Frozen brain sections (10 μm) or cultured neurons were double-labeled with anti-TRIM31 Ab (Sigma, 1:100 dilution) and one of the following Abs: anti-NeuN clone A60 (Millipore, 1:200 dilution), or anti-TIGAR (Santa Cruz, 1:100 dilution). After overnight incubation at 4 °C, secondary Abs including Alexa Fluor 488 donkey anti-rabbit IgG Ab (1:200 dilution; Invitrogen, Gaithersburg, MD, USA) or 595 rabbit anti-Mouse IgG Ab (1:200 dilution; Invitrogen, Gaithersburg, MD, USA) was added for 2 h at room temperature. After rinsing three times, the sections were incubated with a solution containing 200 mg/ml of DAPI (Beyotime, Haimen, China) for nuclear staining.