Pecific siRNA (Santa Cruz). After transfection for 6 h, the transfection medium was replaced with complete medium and cells were incubated for a further 42 h before cell lysis and Western blot. Knockdown experiments were MedChemExpress 86168-78-7 performed in duplicate on three separate occasions.Protein extraction and Western blotProteins from different organs and brain regions were extracted with RIPA buffer (Beyotime, Shanghai, China) containing a protease inhibitor cocktail (Sigma). Tissue lysates were separated by SDS-PAGE and transferred to a PVDF membrane. After 2 h of blocking in 5 11967625 nonfat milk, blots were incubated overnight in primary antibodies (Nischarin, 1:1000, BD Biosciences, San Jose, CA, USA; Integrin a5, 1:1000, Cell Signaling Technology, Danvers, MA, USA; GAPDH, 1:500, Good Here, Hangzhou, China) at 4uC, washed in TBS-Tween 20, and incubated in secondary antibodies for 2 h at room temperature. The protein bands were visualized by enhanced chemiluminescence reagents (Amersham, Arlington Heights, IL, USA) and were exposed to Xray film. Quantification of band intensity was performed using NIH ImageJ software.Cell Proliferation AssayThe effect of Nischarin on the growth of Neuro-2a cells was determined using the MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay as previously described [18]. Briefly, cells were seeded in 200 ml DMEM on 96-well plates (16105/well), and transiently transfected with Nischarin siRNA or control siRNA. At 0, 12, 24 and 48 h after transfection, 100 ml MTT (5 mg/ml, Sigma) was added respectively. After a 4-h incubation at 37uC, 100 ml dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. Cell viability was determined by measuring the absorbence at 570 nm using a microplate reader (Bio-Rad).Wound-healing scratch assaysScratch assays were performed as previously described [19]. Briefly, PC-12 cells were seeded in 6-well plates at 56105 cells/ml and transiently transfected with Nischarin siRNA or control siRNA. At 48 h after transfection, a scratch was made down the center of each well using a plastic pipette tip. Along the scratch line, the detached cells and debris were gently washed away with PBS and the medium was replaced with serum-free culture medium. Cell migration was monitored every 12 h for 48 h under an inverted phase-contrast microscope (Nikon). At each time point, the cell migration edge was traced, and the distance to the initial scratch edge was measured using ImageJ. For each well, three different fields along the scratch were analyzed in triplicate. Motility was calculated as the percentage of the cell migration distance with respect to the initial scratch distance.Single-staining in sectionsRats were 256373-96-3 site anesthetized and transcardially perfused with physiological saline, followed by 4 paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were post-fixed in 4 paraformaldehyde for 1 day, and kept in sucrose solutions of increasing concentration (10, 20 and 30 ) in phosphate-buffered saline (PBS) until they sank. Sections (40 mm) were prepared on a cryostat (Thermo Scientific Microm HM550), blocked with 10 goat serum for 2 h, incubated with primary antibody (mouse antiNischarin monoclonal antibody, 1:100) overnight at 4uC, and then incubated for 2 h with secondary antibody anti-mouse FITC (1:100) at room temperature, washing with PBS after each step. Negative-control staining was performed by the same procedure with the exception of primary antibody incubation. The f.Pecific siRNA (Santa Cruz). After transfection for 6 h, the transfection medium was replaced with complete medium and cells were incubated for a further 42 h before cell lysis and Western blot. Knockdown experiments were performed in duplicate on three separate occasions.Protein extraction and Western blotProteins from different organs and brain regions were extracted with RIPA buffer (Beyotime, Shanghai, China) containing a protease inhibitor cocktail (Sigma). Tissue lysates were separated by SDS-PAGE and transferred to a PVDF membrane. After 2 h of blocking in 5 11967625 nonfat milk, blots were incubated overnight in primary antibodies (Nischarin, 1:1000, BD Biosciences, San Jose, CA, USA; Integrin a5, 1:1000, Cell Signaling Technology, Danvers, MA, USA; GAPDH, 1:500, Good Here, Hangzhou, China) at 4uC, washed in TBS-Tween 20, and incubated in secondary antibodies for 2 h at room temperature. The protein bands were visualized by enhanced chemiluminescence reagents (Amersham, Arlington Heights, IL, USA) and were exposed to Xray film. Quantification of band intensity was performed using NIH ImageJ software.Cell Proliferation AssayThe effect of Nischarin on the growth of Neuro-2a cells was determined using the MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay as previously described [18]. Briefly, cells were seeded in 200 ml DMEM on 96-well plates (16105/well), and transiently transfected with Nischarin siRNA or control siRNA. At 0, 12, 24 and 48 h after transfection, 100 ml MTT (5 mg/ml, Sigma) was added respectively. After a 4-h incubation at 37uC, 100 ml dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. Cell viability was determined by measuring the absorbence at 570 nm using a microplate reader (Bio-Rad).Wound-healing scratch assaysScratch assays were performed as previously described [19]. Briefly, PC-12 cells were seeded in 6-well plates at 56105 cells/ml and transiently transfected with Nischarin siRNA or control siRNA. At 48 h after transfection, a scratch was made down the center of each well using a plastic pipette tip. Along the scratch line, the detached cells and debris were gently washed away with PBS and the medium was replaced with serum-free culture medium. Cell migration was monitored every 12 h for 48 h under an inverted phase-contrast microscope (Nikon). At each time point, the cell migration edge was traced, and the distance to the initial scratch edge was measured using ImageJ. For each well, three different fields along the scratch were analyzed in triplicate. Motility was calculated as the percentage of the cell migration distance with respect to the initial scratch distance.Single-staining in sectionsRats were anesthetized and transcardially perfused with physiological saline, followed by 4 paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were post-fixed in 4 paraformaldehyde for 1 day, and kept in sucrose solutions of increasing concentration (10, 20 and 30 ) in phosphate-buffered saline (PBS) until they sank. Sections (40 mm) were prepared on a cryostat (Thermo Scientific Microm HM550), blocked with 10 goat serum for 2 h, incubated with primary antibody (mouse antiNischarin monoclonal antibody, 1:100) overnight at 4uC, and then incubated for 2 h with secondary antibody anti-mouse FITC (1:100) at room temperature, washing with PBS after each step. Negative-control staining was performed by the same procedure with the exception of primary antibody incubation. The f.
