Mouse anti gapdh 60004 1 ig

The 3,3’-Diindolylmethane (D129118-100 g) and 5-Fluorouracil (F100149-25 g) were purchased from Aladdin (Shanghai, China). Corn oil (S50856) was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). DMSO (Q6949) was from MP Biomedicals (Santa Ana, CA, USA). Mouse anti-GAPDH (60004-1-Ig) was from Proteintech Group (Wuhan, China). Rabbit anti-DHODH (14877-1-AP) was obtained from Proteintech Group (Wuhan, China).

Protein extraction was performed on BV2 and mouse brain samples using RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) supplemented with a 1 mM protease inhibitor cocktail (B14001, Selleck, Houston, TX, USA). The lysates were then centrifuged to gather supernatants. Nuclear and cytoplasmic proteins were extracted using a cytoplasmic and nuclear protein extraction kit (AR0106, Boster, Wuhan, China). The proteins were transferred onto a PVDF membrane (Millipore, Shanghai, China) after being resolved using suitable SDS-PAGE. The membrane was blocked in 5% non-fat milk solution for one hour at room temperature, then incubated overnight at 4 °C with primary antibodies. Immunoblotting was identified using horseradish peroxidase-linked anti-rabbit or anti-mouse secondary antibodies, in conjunction with the BeyoECL Moon super sensitivity detection kit (Beyotime, Shanghai, China). The antibodies and concentrations used for Western blotting were as follows: rabbit anti-TonEBP (ab3446, Abcam, Waltham, USA) 1:1000, rabbit anti-NF-κB p65 (8242, Cell Signaling Technology, Danvers, MA, USA) 1:2000, rabbit anti-phospho-NF-κB p65 (Ser536) (3033, Cell Signaling Technology, Danvers, USA) 1:500, mouse anti-PELI1 (TA807629S, ORIGENE, Rockville, MD, USA) 1:2000, and mouse anti-GAPDH (60004-1-Ig, Proteintech, Wuhan, China) 1:50,000.

Proteins were separated via 10% or 15% Tris-glycine SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were probed with rabbit anti-PSD95 (3450 Cell Signaling, MA, USA), mouse anti-DcR3 (33302, Biolegend, CA, USA), rabbit anti-syndecan-1 (10593–1-AP, ProteinTech, IL, USA), rabbit anti-glypican-1 (sc-66910, Santa Cruz biotechnology, TX, USA), mouse anti-Aβ (6E10, SIG-39320, COVANCE, NJ, USA), anti-YM1 (01404, Stem Cell technology, Vancouver, Canada), mouse anti-GAPDH (60004-1-Ig, ProteinTech, IL, USA), and mouse anti-actin (MAB1501, Millipore, MA, USA) antibodies. The membranes were washed and probed with the HRP-conjugated goat anti-mouse IgG and goat anti-rabbit IgG (12–349, AP132P, Millipore, MA, USA). Protein signals were developed by using a chemiluminescent substrate ECL detection system (WBKLS0500, Millipore, MA, USA) and quantified by using a luminescence imaging system (LAS-4000, Fujifilm, Japan).

The following antibodies were used: Rabbit anti-TIM-4 (HPA015625, 1:1000 for WB, Sigma-Aldrich), Rabbit anti-E-cadherin (proteintech, 20874-1-AP, 1:1000), Rabbit anti-N-cadherin (proteintech, 22018-1-AP, 1:1000), Rabbit anti-vimentin (proteintech, 10366-1-AP, 1:1000), Mouse anti-HA tag (M180-3, 1:5000 for WB, MBL), Mouse anti-GAPDH (60004-1-Ig, 1:5000 for WB, ProteinTech).

HCT-8 cells were lysed in RIPA buffer (25 mM Tris-HCl, pH = 7.4; 150 mM NaCl; 1% NP-40; 0.5% Na-deoxycholate) supplemented with protease inhibitor cocktail. Proteins were separated by 10–12% SDS–PAGE, blotted onto PVDF membrane and incubated with primary antibodies (mouse anti‐GAPDH, 60004-1-Ig, Proteintech, 1:5000; rabbit anti-NOP53, CST, 1:1000; rabbit anti-p53, 10442-1-AP, Proteintech, 1:1000; rabbit anti-p21, 10355-1-AP, Proteintech, 1:1000) overnight at 4 °C, followed by incubation with appropriate secondary antibody (Goat Anti-Rabbit IgG(H + L) HRP, GAR0072, MULTISCIENCES and Goat Anti-Mouse IgG(H + L) HRP, GAM0072, MULTISCIENCES, Hangzhou, China, 1:5000) for 1 h at RT. The protein intensity was analyzed using Image Lab v5.2.1.