Affinipure goat anti rabbit igg h l

Proteins were isolated from non-activated and activated mouse PaSCs 48 h after transfection with either Notch3 siRNA or control siRNA using radioimmunoprecipitation assay buffer (Beyotime Bio, Haimen, China) containing a protease inhibitor cocktail and PMSF. Cells were centrifuged at 12,000 g for 30 min, and supernatant fractions were collected. Protein was measured with a Pierce™ BCA protein assay kit according to the manufacturer’s instructions (Prod #23225, Thermo Scientific, Waltham, USA). Equal amounts of protein were loaded and separated on 8% or 10% PAGE gels and transferred onto nitrocellulose filter membranes (Millipore, Darmstadt, Germany). The membranes were incubated with 5% milk for 1 h at room temperature and then probed with primary antibodies overnight at 4 °C. The primary antibodies are shown in Table 1. Subsequently, the membranes were incubated with peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H + L) (ZB-2301, ZSGB-Bio) for 1 h at room temperature. The proteins were detected with enhanced chemiluminescence (Millipore, Darmstadt, Germany) using an LAS3000 System (Fujifilm, Japan). Protein levels were normalized to GAPDH levels and quantified using ImageJ software (NIH).