System geldoc xr imagelab

Fresh lumbar spinal cord tissue was extracted and put into RIPA lysate (AR0102-100, Boster), adding protease inhibitor PMSF(AR1178, Boster) and phosphatase inhibitor (AR1183, Boster). BCA protein concentration assay kit (AR0146, Boster) was used to determine the protein concentration.
The total proteins from the spinal cord were separated by electrophoresis using 10% SDS–polyacrylamide gel, then transferred onto PVDF membranes. The membranes were blocked with 5% non-fat milk for 2 h at room temperature, then incubated overnight at 4 °C with the following antibodies: Rabbit Anti-SHH Polyclonal Antibody (bs-1544R, Bioss), Rabbit Anti-Gli-1 Polyclonal Antibody (bs-1206R, Bioss), Phospho-AKT (Ser473, Cell Signaling Technology), AKT polyclonal antibody (AP0095, Bioworld) or β-actin (AP0060, Bioworld) antibodies. After washing, the membranes were incubated with Goat anti-Rabbit IgG (BA1054, Boster) for 1 h at room temperature. The blots were visualized by the chemiluminescence-based detection kit (ECL Kit, Boster). Densitometry analysis was performed with a System Gel Doc XR + IMAGE LAB (Bio-Rad, USA) and quantified with NIH Image software (Image J).

Cells were seeded into 60 mm culture plate. After 48 hours, cells were harvested when cell confluence reached to 90%. Cell lysate were separated with 15% SDS-PAGE, transferred to nitrocellulose and probed with hTRIR polyclonal antibody. Luminescent signals were detected with SYSTEM GelDoc XR + IMAGELAB (Bio-Rad).

T7 transcribed RNA, total cellular RNA or RNA oligos were incubated with different amount of purified hTRIR in digestion buffer (25 mM NaCl, 0.5 mM Tris pH 7.5, 2% (v/v) glycerol) at 37 °C for 30 min or indicated time. RNAs were harvested and separated by 7 M urea polyacrylamide gel electrophoresis (PAGE) or 1.5% agarose (Biowest) gel. T7 Transcribed RNA signals were detected by Typhoon cyclone (PerkinElmer). Total cellular RNA signals were detected by SYSTEM GelDoc XR+ IMAGELAB (Bio-Rad). Small RNA was stained with stain-all (Sigma) and scanned with Epson scanner.