Super signal west dura detection reagent

Proteins were isolated 72 h after siRNA transfection using cell lysis buffer (50 mM Tris/HCl, pH 7.2, 150 mM NaCl, 5 mM NaF, 0.25 mM EDTA, 1% Triton-X-100, 1% SDS, 1 mM NaVO₄, 5 μg/ml pepstatin, 5 μg/ml leupeptin, 0.14 U/ml aprotinin). Protein concentrations were determined by Bradford analysis, and lysates containing 30 μg total protein were mixed with Laemmli buffer, boiled, separated by 4–12% Bis-Tris gel electrophoresis (Nupage, NP0335) and blotted onto a polyvinylidene fluoride membrane. Membranes were blocked in 3% milk powder in Tris-buffered saline with Tween (TBS-Tween). Specific protein bands were detected using primary and secondary antibodies (listed in Table 3) and visualized bioluminescence using the Super Signal^® West Dura detection reagent (Thermo Fisher Scientific, Waltham, MA, United States) and a CCD camera (Raytest, Straubenhardt, Germany). Each immunoblot shown is a representative example out of at least two independent biological replicates (n > 2). Uncropped images of the blots are included as ded as Supplementary Material S1.

Proteins were isolated using cell lysis buffer (50 mM Tris/HCl pH 7.2, 150 mM NaCl, 5 mM NaF, 0.25 mM EDTA, 1% Triton-X-100, 1% SDS, 1 mM NaVO₄, 5 μg/ml pepstatin, 5 μg/ml leupeptin, 0.14 U/ml aprotinin). After a Bradford analysis, 30 μg of lysates were supplemented with Laemmli buffer, boiled, separated in a 10–15% SDS gel and blotted to a polyvinylidene fluoride membrane. Protein bands were visualized using primary and secondary antibodies (Additional file 1: Table S3), the Super Signal® West Dura detection reagent (Thermo), and a CCD camera (Raytest, Straubenhardt, Germany). Each immunoblot shown is a representative example out of a minimum of three independent biological replicates (n > 3). Where indicated, a quantitative analysis of band signals was performed using the software XStella.