Anti notch3 ab23426

Bone marrow stromal cells from control or BGLAP-Cre;Rosa^Notch3 mice were extracted in buffer containing 25 mm Tris-HCl (pH 7.5), 150 mm NaCl, 5% glycerol, 1 mm EDTA, 0.5% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, and a protease inhibitor mixture (all from Sigma-Aldrich). Quantified total cell lysates (35 μg of total protein) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in 8% or 10% polyacrylamide gels and transferred to Immobilon-P membranes (Millipore). The blots were probed with anti-NOTCH3 (ab23426) (Abcam) and anti-β-Actin (3700) antibodies (Cell Signaling Technology) and exposed to anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase (Sigma-Aldrich) and incubated with a chemiluminescence detection reagent (Bio-Rad). Chemiluminescence was detected on a ChemiDoc XSR+ molecular imager (Bio-Rad) with Image Lab software (version 6.0.1), and the amount of protein in individual bands was quantified (49 (link)).

Cells were lysed in RIPA lysis buffer (P0013C; Beyotime Co.). Cell extracts were collected by centrifugation, analyzed by 12% or 10% SDS/PAGE, and then followed by western blotting using mouse monoclonal anti‐GPX4 (67763‐1‐Ig; ProteinTech Group, Wuhan, China), rabbit polyclonal anti‐Notch3 (ab23426; Abcam, Cambridge, UK), and rabbit polyclonal anti‐PRDX6(13585‐1‐AP; ProteinTech Group). After the primary incubation, membranes were incubated with secondary horseradish peroxidase (HRP)‐conjugated goat anti‐rabbit or anti‐mouse IgG (A0279 or A0288; Beyotime Co.). Protein bands were visualized using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA). The integrated optical density of band was analyzed using imagej software (National institutes of health, Bethesda, MD, USA).