Ab252426

Whole cell extracts of MC3TE-E1 for western blotting were prepared after the indicated treatment for 3 days as previously described [19 (link)]. 20 μg protein from total cell lysate was prepared using PRO-PREPTM protein extraction solution (Boca Scientific Inc., Boca Raton, FL) and carried out subsequent electrophoresis, according to the manufacturer’s instruction. The primary antibodies against the following proteins: FoxO1 (Abcam, ab52857, 1:1000), SIRT1 (Abcam, ab189494, 1:1000), human catalase (CAT, Abcam, ab130029, 1:1000), glutathione peroxidase (GPX1, Abcam, ab108427, 1:1000), superoxide dismutase 2 (SOD2, Abcam, ab252426, 1:1000). Expression levels of the target protein were normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Boster, Wuhan, China, 1:2000) levels in each sample. The following day, HRP-conjugated goat anti-rabbit, which used as secondary antibodies, was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and the results were detection and analysis by using an iBrightCL1000 (Invitrogen, Carlsbad, CA).

A polyclonal rabbit anti-hepcidin 25 (Abcam, ab30760), recognising a 2.8 kDa protein [53 (link)], monoclonal antibody (MAB) human anti-ferritin light chain (Abcam, ab69090), human anti-ferritin heavy chain (Abcam, ab65080), anti-DMT1 (Abcam, ab123085), anti-FPN (Abcam, ab85370), anti-CD42b (Abcam, ab104704), anti-rabbit polyclonal glycophorin (Abcam, ab196568), anti-GFAP (Abcam, ab48050), anti-CD68 (Sigma-Aldrich, AMAB98073), MAB anti-CD11b (Thermo Fisher, Waltham, MA, USA, mAbM1/70), GFAP (Abcam, ab48050), MAB anti-Iba1 (Thermo Fisher, MAB M1/70), polyclonal anti-Iba1 (Wako, Fujifilm, Tokyo, Japan, catalogue number 019-19741), MAB anti-IL-6 (Thermo Fisher, catalog number M620), MAB IL-1β (Thermo Fisher, ILB1-H67), MAB anti-β amyloid 42 (Covance, Princeton, NJ, USA, SIG 39320), anti-phospho-tau (AT8, Thermo Fisher, MN1020), SOD1 (Abcam, ab252426), S100β (Abcam, ab218514), RUNX1 (Sigma-Aldrich, HPA004176) and OLIG2 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-365644) were used for IHC or Western blotting. The following secondary antibodies were used: biotinylated goat anti-rabbit-Ig and biotinylated horse anti-mouse (both from Vector Laboratories, 1:250 for IHC), Alexa Fluor 568-labelled donkey anti-mouse-Ig, Alexa Fluor 488-labelled donkey anti-rabbit-Ig and Alexa Fluor 568-labelled donkey anti-goat-Ig (all from Invitrogen, 1:1000 for immunofluoresence).

Protein samples were prepared from human serum (DS and age-matched controls, n = 20). Twenty μg protein samples were separated on 4–12% Nu-PAGE Bis-tris (Bis (2-hydroxyethyl)-amino-tris (hydroxymethyl)-methane) gradient gels and transferred to 0.2 μm pore size PVDF for Aβ42, hepcidin and S100β, and 0.45 μm pore size PVDF membranes for SOD1, FTL, FPN, TREM2 and albumin using the NuPAGE electrophoresis system (Invitrogen). Membranes were incubated with different antibodies, i.e., anti-β amyloid (Covance, SIG 39320), SOD1 (Abcam, Cambridge, UK, ab252426), S100β (Abcam, ab218514), anti-ferritin light chain (FTL, Abcam, ab69090), anti-FPN (Abcam, ab85370), anti-hepcidin 25 (Abcam, ab30760), anti-TREM2 (Abcam, ab86491) and anti-albumin (Abcam, ab10241) antibody, in blocking buffer for 24 h at 4 °C, and then washed three times with 0.1 M Tris saline buffer containing 1% Tween 20 (TBST), followed by incubation for 1 h at RT with HRP-conjugated secondary antibodies, anti-rabbit IgG (1:3000, DAKO) or anti-mouse IgG (1:3000; DAKO) antibodies. Binding was detected with ECL Plus chemiluminescence reagents.