Clarity western ecl substrate chemiluminescence detection kit

Proteins were extracted from TC32 and A673 ES cell lines in RIPA buffer (150 mM NaCl, 1% (v/v) NP40, 50 mM Tris-HCl pH 8.0, 0.1% (v/v) SDS, 1mM EDTA, and 0.5% (w/v) deoxycholate) supplemented with protease inhibitor, 10 mM NaF and 2 mM NaOv. Immunoblotting was performed using the following antibodies: EWS-FLI1 expression was determined using the anti-FLI1 antibody (C-19) (Santa Cruz, #sc-356) overnight at 1:1000 dilution, followed by anti-rabbit IgG, HRP (Cell Signaling, #7074) for 1h at 1:10000; and calnexin (E-10) (Santa Cruz, #sc-46669) overnight at 1:1000 dilution, followed by anti-mouse IgG-HRP (Cell Signaling, #7076) for 1h at 1:10000. Protein bands were visualized using the Clarity Western ECL Substrate chemiluminescence detection kit (Bio-Rad, #170-5060). ImageJ software was applied for densitometric quantifications.

WB analysis was carried out as described by Nutzmann et al. [71 (link)]. Total protein (60 μg) was separated by SDS-PAGE and then transferred to PVDF membrane using a wet blotting system (Bio-Rad). The membrane was blocked with 5% BSA, incubated overnight with primary antibody (BxlB) and then gently shaken at room temperature following the addition of immunoglobulin G anti-rabbit secondary antibody labeled with peroxidase. Protein detection was carried out using the Clarity Western ECL Substrate chemiluminescence detection kit (Bio-Rad), as described by the manufacturer.

Proteins were extracted from kidney and WT frozen tissues in RIPA buffer (150 mM NaCl, 1% (v/v) NP40, 50 mM Tris-HCl pH 8.0, 0.1% (v/v) SDS, 1 mM EDTA and 0.5% (w/v) deoxycholate) supplemented with 10 mM NaF and 2 mM NaOv. Samples were incubated for 20 min on ice and centrifuged for 15 min at 13000 r.p.m. at 4°C. Supernatants were collected and quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific Inc.). Equivalent amounts of proteins were resolved by SDS polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Darmstadt, Germany). Immunoblotting was performed using the following antibodies: anti-ABCB4 (Abcam-ab184878) overnight at 1:200 dilution; anti-MRP1 (Abcam-ab137406) overnight at 1:200 dilution; anti-β-actin (A5441 clone AC-15) for 1.5 h at 1:10000 dilution; anti-rabbit IgG, HRP (Cell Signaling, ref#7074) for 1 h at 1:10000; anti-mouse IgG-HRP (Cell Signaling, ref#7076) for 1 h at 1:10000. Protein bands were visualized using the Clarity Western ECL Substrate chemiluminescence detection kit (Bio-Rad, ref#170-5060). All of the antibodies were previously analyzed for antigen specificity in our laboratory, all conditions being optimized for specific antigen detection, with elimination of nonspecific reactivity.