Chemidoc xsr molecular imager

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 from control and experimental 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 sodium orthovanadate, 10 mM NaF, 1 mM phenyl methyl sulfonyl fluoride, and a protease inhibitor cocktail (all from Sigma–Aldrich). Total cell lysates (40 μg of total protein) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 8 or 12% polyacrylamide gels and transferred to Immobilon-P membranes (Millipore). The blots were probed with anti-HES1 (11988) and β-actin (3700) antibodies from Cell Signaling Technology or anti-NFATc1 antibody (556602) from BD Biosciences. The blots were exposed to anti-rabbit, antirat, or antimouse IgG conjugated to horseradish peroxidase (Sigma–Aldrich) and incubated with a chemiluminescence detection reagent (Bio-Rad). Chemiluminescence was detected by ChemiDoc XSR+ molecular imager (Bio-Rad) with Image Lab software (version 5.2.1), and the amount of protein present in individual bands was quantified (25 (link)).