Fusion fx imager fusion software

Western blotting was carried out as previously described [26 (link)]. Briefly, RIPA buffer (R0278, Sigma-Aldrich) with a protease inhibitor cocktail (complete Tablets Mini, Roche, Basel, Switzerland) was used to isolate total protein from cells. A colorimetric test (PierceTM BCA Protein Assay Kit, Thermo Fisher Scientific) was used to determine the total protein concentration. SDS-PAGE was used to separate 10 µg of total protein under reducing conditions, and immunoblot analyses were carried out with antibodies (Supplementary Table S1) against PAX6 (clone#poly901301—1:1000; clone#D3A9V—1:1000) and GAPDH (1:50,000), followed by horseradish peroxidase-labeled anti-mouse or rabbit IgG (Jackson ImmunoResearch Europe, Ely, UK). Enhanced chemiluminescence Western blot detection reagent (GE Healthcare, München, Germany) and FUSION FX imager/fusion software (Vilber Lourmat, Collégien, France) were used to visualize protein bands.

Cells were lysed in ice cold RIPA buffer and the insoluble material was removed by centrifugation at 12,000 g, 10 min. Whole cell lysates were separated on 10% SDS-polyacrylamide gels and electroblotted onto nitrocellulose membranes (Amersham Protran, GE Healthcare). Membranes were blocked in 5% (weight/volume) nonfat milk in TBS-T (20 mM Tris base, 137 mM sodium chloride, 0.1% Tween 20, pH 7.6) for 1 h. Western blots were performed using rabbit anti Caspase 3 antibodies (Cell Signaling, #9662, 1:1,000), which detect both cleaved and uncleaved forms of caspase 3, and mouse anti α-tubulin antibodies (Abcam, #ab7291, 1:10,000). Secondary antibodies conjugated with horseradish peroxidase were from Jackson ImmunoResearch (#111-036-047 and #115-036-062, 1:10,000). Protein bands were visualized using the enhanced chemiluminescence Western blot detection reagent (GE Healthcare) and the Fusion Fx Imager/Fusion software (Vilber Lourmat). Densitometry analysis of protein bands was carried out using the Bio-ID software (Vilber Lourmat). For quantification of Western blots, the levels of cleaved caspase 3 were normalized to the levels of uncleaved caspase 3.