Peroxidase conjugated anti rabbit igg

To confirm the effect of IL-4 on proliferation of NSCs and to investigate its the mechanism, the expression of proliferating cell nuclear antigen (PCNA) using western blot analysis. Cells were lysed in a buffer containing 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 140 mM NaCl, 1% (w/v) Nonidet P-40, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, and 10 μg/ml aprotinin. Cell lysates were separated by 12% SDSpolyacrylamide gel electrophoresis, and electrotransferred to a polyvinylidene difluoride membrane (Bio-Rad). The membranes were incubated with blocking buffer (1× Tris-buffered saline, 1% BSA, 1% nonfat dry milk) for 1 h and then incubated with the primary antibody against PCNA (Santa Cruz Biotechnology; 1:1,000) for overnight at 4°C. Primary antibodies were visualized with peroxidase-conjugated anti-rabbit IgG and a chemiluminescent detection system (Santa Cruz Biotechnology, Inc., USA). The ChemicDoc XRS system (Bio-Rad) was used for visualization and quantification using Quantity One imaging software (Bio-Rad).

Cells were harvested, washed with PBS, and resuspended in lysis buffer (50 mmol/L Tris‐HCl [pH 7.6], 150 mmol/L NaCl, 1 mmol/L PMSF, 1 mmol/L DTT, 10 µg/mL aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin A, and 1% NP‐40) with phosphatase inhibitor (10 mmol/L NaF and 1 mmol/L Na₃VO₄) for 20 minutes on ice. After centrifugation, the supernatants were isolated and used as cell lysates. The cell lysates were separated by SDS‐PAGE and transferred onto PVDF membranes. The membranes were blocked with blocking buffer (0.1% casein, 0.1% gelatin, and 0.1% Tween‐20 in TBS) and incubated with the following primary Abs: rabbit anti‐DYRK2 (Sigma‐Aldrich), mouse anti‐p53, c‐Myc, Cyclin D1 and GAPDH and rabbit anti‐Cyclin D2 (Santa Cruz Biotechnology), mouse anti‐phospho‐p53‐Ser46 (Bio Academia), mouse anti‐cleaved PARP, and rabbit anti‐cleaved caspase 3 (Cell Signaling Technology). Membranes were then washed three times in TBS with 0.05% Tween‐20, and incubated with peroxidase‐conjugated anti‐rabbit IgG (Santa Cruz Biotechnology) or peroxidase‐conjugated anti‐mouse IgG κ‐BP (Santa Cruz Biotechnology). Signals were detected using a chemiluminescent regent, ImmunoStar LD (Wako). Signals were observed and band intensity was measured using a Fusion‐Solo system (M and S Instruments).

The Rabbit polyclonal ISOC1 antibody anti-human ISOC1 was purchased from Abcam (#ab118245; Cambridge, UK). Anti-GAPDH (#sc-32233), peroxidase-conjugated antirabbit IgG (#sc-2004) and anti-mouse IgG (#sc-2005) were purchased from Santa-Cruz Biotechnology, Inc. (Dallas, TX, USA). RPMI 1640 medium with L-Gultamine (#11875-093), fetal bovine serum (FBS; #10100-147) and Lipofectamine^® 2000 (#11668019) were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Penicillin-streptomycin (#1107440001) and Giemsa staining solution (#32884) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany).

Western blots were performed as described in a previous report.^{24 (link)} Nine-week-old male mice of wild type and STEP^C230X−/− were used in this assay. Briefly, the brain was quickly removed and homogenized in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS and 0.1% sodium deoxycholate) containing a cocktail of protease inhibitors (Roche, Mannheim, Germany). Protein samples were resolved on 10% SDS-polyacrylamide gel electrophoresis; the transferred blots were incubated with primary antibodies, followed by secondary antibody, and the signals were visualized using an ECL kit (Amersham Bioscience, Little Chalfont, UK). Antibodies used were monoclonal anti-STEP (23E5, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-STEP (Ser221/Ser49, Millipore, Billerica, MA, USA), anti-ERK1/2 (Cell Signaling Technology, MA, USA), anti-phospho-ERK1/2 (Thr202/Tyr204, Cell Signaling Technology), anti-μ-opioid receptor (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-δ-opioid receptor (Santa Cruz Biotechnology), anti-κ-opioid receptor (GeneTex, Irvine, CA, USA) and anti-actin (Millipore). The secondary antibodies used were peroxidase-conjugated anti-rabbit-IgG and anti-mouse-IgG (Santa Cruz Biotechnology).