Ab31828

Total protein was extracted from brain tissues using RIPA (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) containing PMSF (0.1 mM). The protein concentration was determined using BCA kit (Thermo Fisher Scientific, Inc.). The sample was mixed with the loading buffer and heated at 100˚C in a water bath for 10 min. Proteins (50 µg) were separated by 10% SDS-PAGE and transferred onto a PVDF membrane (cat. no. ISEQ00010; EMD Millipore). Membranes were blocked using 5% skim milk at 4˚C for 2 h and washed with 0.1% TBST. Membranes were incubated with primary antibodies against phosphorylated (p) p38MAPK (ab31828; 1:1,000; Abcam), MAPK1 (ab31828; 1:5,000; Abcam), iNOS (ab213987; 1:5,000; Abcam) and GAPDH (ab22555; 1:2,000; Abcam) overnight at 4˚C. After three washes with TBST for 6 min, membranes were incubated with the secondary HRP-labeled goat anti-rabbit IgG antibody (TA140003; 1:5,000; OriGene Technologies, Inc.) at room temperature for 2 h. Membranes were washed three times with TBST for 6 min and placed into TBS. Enhanced chemiluminescence reagent (BB-3501; Cytiva) was used to detect the signal on the membrane. Images were acquired on a Bio-Rad image analysis system (Bio-Rad Laboratories, Inc.). The data were analyzed via densitometry using ImageJ software V2.1.4.7 (National Institutes of Health) and normalized to expression of the internal control GAPDH.

Hippocampal cells and cerebrospinal fluid were obtained as described previously (34 (link)), homogenized in lysate buffer containing protease inhibitor and centrifuged at 8,000 g/min at 4°C for 10 min. Total protein was extracted from the resulting supernatant using a Protein Extraction kit (20021; Qiagen Sciences, Inc., Gaithersburg, MD, USA), according to the manufacturer's instructions. SDS assays were performed as described previously (35 (link)). For western blot analysis, primary antibodies anti-bax (1:1,000; ab32503), anti-Bcl-2 (1:1,000; ab194583), anti-caspase-3 (1:1,000; ab13847), anti-caspase-9 (1:1,000; ab18571), anti-p38 (1:1,000; ab31828), anti-ERK (1:1,000; ab176660), anti β-actin (1:1,000; ab8226; all Abcam; Cambridge, UK) were added to PVDF membranes (EMD Millipore) and incubated at 4°C overnight. Membranes were blocked in 5% skimmed milk for 1 h at 37°C and then incubated with HRP-conjugated goat anti-rabbit IgG mAb (PV-6001; ZSGB-BIO) for 24 h at 4°C. A Ventana Benchmark automated staining system was used to determine protein expression in tumor tissues (Olympus BX51; Olympus Corporation).