Ecl plus chemiluminescence reagent

The cells were collected and homogenized in lysis buffer (50 mM Tris-HCl pH 7.4, 5 mM EDTA, 250 mM NaCl, and 0.1% Triton x-100) supplemented with protease and phosphatase inhibitors and quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific by Life Technologies, Carlsbad, CA, USA). An amount of 20–40 μg of protein extracts from each sample were denatured in 5× Laemmli sample buffer and subjected to 7.5% precast TGX Stain-Free polyacrylamide gel (Bio-Rad Laboratories, Munchen, Germany) electrophoresis. Proteins were electrotransferred onto nitrocellulose membrane and blocked with 1× Phosphate Buffered Saline (PBS) with 1% casein (Bio-Rad Laboratories, Munchen, Germany) for 40 min at room temperature. The immunoblots were performed using anti-β-actin, anti-c-MYC, anti-p38α MAPK, anti-MEK, anti-phospho-MAPKAPK-2 (Thr334) (p-MK2), anti-Ubiquitin, anti- β-catenin, anti-cyclin D1, anti-LC3, anti-cleaved PARP, and anti-phospho-p44/42 MAPK (Thr202/Tyr204) (all from Cell Signaling, Danvers, MA, USA). After incubation with the HRPO-conjugated secondary antibodies (GE Healthcare, Milwaukee, WI, USA), the signal was revealed using the ECL-plus chemiluminescence reagent (GE Healthcare, Milwaukee, WI, USA) according to the manufacturer’s instructions. Densitometric evaluations were carried out using the ImageJ and ImageLab software.

In the experiments for studying protein secretion, medium harvested from empty vector, R5-6, or R5-6C transfected cells was mixed with methanol to a final concentration of 60%, and incubated at -20°C for 1 h, followed by centrifugation at 16000 x g for 2 min at 4°C. The pellet was resuspended in urea loading buffer (9 M urea, 125 mM Tris-HCl, pH 6.8, 715 mM β-mercaptoethanol, 2% SDS). In the experiments for studying the effect of reelin subregions on protein expression, RAW 264.7 cells were equilibrated in DMEM for 3 h, then incubated with DMEM ± 0.2 μg/ml of purified R5-6, or with control-, R5-6- or R5-6C-conditioned medium for 3 h. In the experiments involving PI3K and Sp1 inhibitors, 50 μM LY294002 or 100 nM mithramycin A were added into the medium. The cells were lysed in M-PER mammalian protein extraction reagent. Cell lysates and the proteins precipitated from the culture medium were resolved on 10% SDS-PAGE gels. Proteins were transferred to a PVDF membrane. After blocking with 3% fat-free milk, membranes were incubated with antibodies as indicated in the figure legends. Immunoreactive bands were visualized using ECL-plus chemiluminescence reagent (GE Healthcare–Amersham) and analyzed with a GS-700 Imaging Densitometer (Bio-Rad) [28 (link)].

Immunoblotting analyses were performed according to Cell Signaling’s instructions. Briefly, cells were homogenized in 1X lysis buffer (50 mM Tris–HCl pH 7.4; 5 mM EDTA; 250 mM NaCl; 0.1% Triton X-100) supplemented with protease and phosphatase inhibitors (1 mM PMSF; 1.5 μM pepstatin A; 2 μM leupeptin; 10 μg/ml aprotinin, 5 mM NaF; 1 mM Na3VO4). 15 to 20 μg of protein extracts from each sample were denatured in 5× Laemmli sample buffer and loaded into an SDS-polyacrylamide gel for western blot analysis. Western blots were performed using anti-β-Actin (Sigma) and anti-FoxO3A (Cell Signaling). Western blots were developed with the ECL-plus chemiluminescence reagent (GE Healthcare) as per manufacturer’s instructions.