Anti gapdh 5174

Cells or ground flash frozen tissue were lysed in 10 mM Tris (pH 7.4), 150 mM sodium chloride, 1 mM EDTA, 1% Nonidet P-40, 1% Triton X-100, 1 mM sodium orthovanadate, 0.2 mM PMSF, and Pierce protease inhibitor mixture. Samples were separated on a 10% gel and transferred onto nitrocellulose membranes (GE Healthcare). Anti-β-tubulin (sc-9104), anti-RelB (sc-226), anti-p65 (sc-372) antibodies (Santa Cruz Biotechnology); anti-GAPDH (5174); and anti-phospho-p65(S536) (3031) antibodies (Cell Signaling) were used. Antigen-antibody complexes were visualized by enhanced chemiluminescence using Immobilon Western blotting kit (Millipore).

Antibodies were from the following sources: Anti-p65 (8242), anti-p-p65 (3033), anti-cleaved caspase-3 (9664), and anti-GAPDH (5174) were from Cell Signaling Technology; Anti-IL-6 (66146) was from Proteintech; Anti- F4/80 was from Servicebio (Wuhan China). The secondary antibody for immunoblot was from Thermo Fisher Scientific. Special regents were shown as follows: digoxigenin-labeled mmu–miR-26a-5p LNA probes and the Fluorescence In Situ Hybridization Kit were from Servicebio; miR-26a-5p mimic, anti-miR-26a-5p, and relative negative control oligonucleotide were from Ribo Biotechnology (Guangzhou, China); LPS was from Sigma, and TPCA-1 (A4602) was from APExBIO.

Hippocampal tissue or HCN-2 cells were homogenized using RIPA lysis buffer (Boster Biotechnology, Wuhan, China) containing phenylmethanesulfonyl fluoride (Boster Biotechnology, Wuhan, China) for 15 min. Protein concentrations were then measured with a BCA kit (Beyotime Biotechnology, Haimen, China). Membranes were incubated overnight at 4°C with the following primary monoclonal antibodies: Anti-Nupr1 (ab6028), anti-Cleaved Caspase-3 (ab49822), anti-Bcl-2 (ab196495) (1: 1000 dilution; Abcam, UK), anti-C/EBPβ (2895), anti-IGFBP5 (10941), anti-Bax (2772), and anti-GAPDH (5174) (1:1000 dilution; Cell Signaling Technology, USA). Membranes were then incubated with horseradish peroxidase-labeled secondary mouse anti-rabbit (93702) or rabbit anti-mouse (5127) (1: 5000 dilution; Cell Signaling Technology, USA) antibodies for 2h. Super Signal West Pico Chemiluminescent Substrates (Pierce Biotechnology, Rockford, IL, USA) and GEL-PRO ANALYZER software (Bio-Rad Laboratories, Hercules, CA, USA) were used to visualize and quantify protein band absorbance.

The following antibodies were used: anti-GAPDH (5174, Cell Signaling Technology), anti-SLC7A11 (26864-1-AP, Proteintech), anti-p53 (10442-1-AP, Proteintech), anti-GPX4 (A11243, ABclonal Technology), anti-ACSL4 (A6826, ABclonal Technology), anti-FTL (ab69090, Abcam), anti-NRF2 (M200-3, Medical Biological Laboratories), anti-ATF3 (P18847, Cusabio), anti-ATF4 (P18848, Cusabio), anti-HA (M180, Medical Biological Laboratories), anti-DDDDK (M185, Medical Biological Laboratories), anti-ub (10201-2-AP, Proteintech), anti-RND2 (GXT56070, GeneTex), anti-RND3 (GTX81316, GeneTex) and anti-Mouse IgG (A7028, Beyotime Biotechnology). Several anti-RND1 antibodies in the market has been tried which showed the poor sensitivity, and they were prone to non-specific reactions with other RND members, such as RND2 and RND3. We selected the latter part of the RND1 sequence (CSLSKRLLHLPSRSE) to predict the antigen peptide and synthesized an anti-RND1 antibody by Genscript (Nanjing, China). The specificity of RND1 antibody was verified (Additional file 1: Fig. S1B). DMSO was purchased from Servicebio (WD2650, Servicebio). The apoptosis inhibitor Z-VAD-FMK (S7023), the ferroptosis inhibitor Ferrostatin-1 (S7243), the necrosis inhibitor Necrosulfonamide (S8251) and the ferroptosis inducer Erastin (S7242) were purchased from Selleck (USA).

Bafilomycin A1 (S1413), cycloheximide (S7418), and MG132 (S2619) were purchased from Selleck Chem (Houston, TX, USA). Anti-STAMBP (sc-271641) and anti-ubiquitin (sc-8017) antibodies were purchased from Santa Cruz Biotechnology (CA, USA). Anti-RAI14 (ab137118), anti-MMP9 (ab137867), and anti-PABPC1 (10970-1-AP) antibodies were purchased from Abcam (USA). Anti-RBM14 (10196-1-AP) and anti-LIMA1 (16639-1-AP) antibodies were purchased from Proteintech Group (Chicago, USA). An anti-ki67 (BS1454) antibody was purchased from Bioworld Technology (Louis Park, MN, USA). The following antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA): anti-K48-ub (12805), anti-HA-tag (3724S), anti-DYKDDDDK (FLAG) tag (14793), E-Cadherin (3195T), Slug (9585T), Vimentin (5741), and anti-GAPDH (5174) antibodies.

Anti-PERK (3192), anti-p-PERK (3179S), anti-CHOP (2895), anti-caspase-4 (4450), anti-cleaved caspase-3 (Cleaved caspase-3) (9664) and anti-GAPDH (5174) antibodies were obtained from Cell Signaling (Danvers, USA). Anti-CHOP (15204-1-AP), anti-78-kDa glucose-regulated protein (GRP78) (66574-1-Ig), anti-Nuclear respiratory factor 2 (Nrf2) (16396-1-AP), anti-Keap1 (60027-1-Ig), anti-GAPDH (10494-1-AP) and anti-β-actin (20536-1-AP) were obtained from Proteintech (Chicago, USA). Anti-apelin (ab125213) and anti-caspase-12 (ab62484) were obtained from Abcam (Cambridge, UK). Anti-β-tubulin (GB11017) was obtained from Servicebio (Wuhan, China). All secondary antibodies were obtained from Abcam (Cambridge, UK). Apelin-13 (A6469, purity ≥ 95.0%) and 4-phenylbutyrate (4-PBA) (P21005) were purchased from Sigma-Aldrich (St. Louis, USA). GSK2656157 (5.04651) was purchased from EMD Millipore (Massachusetts, USA). Tunicamycin (TM) (ab120296) was purchased from Abcam (Cambridge, UK). Iohexol was purchased from GE Healthcare (Shanghai, China).

Antibodies: anti-FIH-1(ab237544) was from Abcam (Cambridge, UK). Anti-cleaved caspase-3(9664) and anti-GAPDH (5174) were from Cell Signaling Technology (Boston, MA). All secondary antibodies for immunoblotting analysis were from Thermo Fisher Scientific (Waltham, MA). FITC -conjugated goat anti-rabbit IgG were from Abcam. Special reagents: Digoxigenin-labeled mmu-miR-122-5p LNA probe (Servicebio, Wuhan, China), Fluorescence in situ hybridization Kit (Servicebio, Wuhan, China), miR-122-5p mimic (Ruibo, Guangzhou, China), anti-miR-122-5p (Ruibo, Guangzhou, China), Lipofectamine 2000 (Thermo Fisher, Waltham, MA).