Immobilon western chemiluminescent hrp substrate wbklso500

Embryos were dissected from pregnant females at gestational stage 8.5~9.5 and the deciduas were torn off. Total proteins were extracted with Mammalian Protein Extraction Reagent (M-PER; Thermo Scientific, Waltham, MA, USA) containing a protease inhibitor cocktail (P8340, Sigma-Aldrich). Protein concentrations were determined with a Pierce BCA protein assay kit (Thermo Scientific). Appropriate amounts of the extracts were fractionated by SDS-PAGE, electrotransferred to PVDF membranes, and detected with antibodies against CUL4B (HPA011880, Sigma-Aldrich), β-catenin (14–6765, ebioscience, San Diego, CA, USA), phospho-β-catenin (S33/S37/T41) (9561, Cell Signaling Technology, Danvers, MA, USA), cyclin D1 (ab10540, Abcam, Cambridge, MA, USA), cyclin D2 (ab3087, Abcam), cyclin D3 (ab28283, Abcam), cyclin E (630701, Biolegend, San Diego, CA, USA), and β-actin (A5060, Sigma-Aldrich). Signals from the reaction with anti-mouse IgG and anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibodies (AP124P and AP132P, Millipore) were developed with the Immobilon Western Chemiluminescent HRP substrate (WBKLSO500, Millipore). The signals were semiquantified using Multi Gauge V3.0 software (Fujfilm) and normalized against that of β-actin.

Testes were collected at P20 mice. Briefly, testes were homogenized with tissue protein extraction reagent (T-per, Thermo Scientific, Waltham, MA, USA) containing 1% protease inhibitor cocktail (P8340, Sigma-Aldrich), according to the manufacturer’s instructions. Protein concentrations were determined using a Pierce BCA protein assay kit (Thermo Fisher Scientific). Protein samples were separated by SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes. Bound proteins were detected using the indicated primary antibodies (see Supplementary Table S1 online), and were visualized using HRP-conjugated secondary antibodies and Immobilon Western Chemiluminescent HRP substrate (WBKLSO500, Millipore). Bands were semiquantified with Multi Gauge V3.0 software (Fujifilm, Tokyo, Japan) and were normalized against α-tubulin.