Western blot method was done accordingly to the previously published protocol [28 ]. In brief, cells were washed with PBS and subsequently lysed in the sample buffer (2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.002% bromphenol blue and 0.06 M Tris HCl, pH 6.8), sonicated and thermally denatured. Electrophoretically separated proteins, in the SDS-PAGE gels, were transferred onto Immobilon-P PVDF Membrane (IPVH00010, Millipore) and detected by primary and corresponding HRP-conjugated secondary antibodies on Fusion SL imaging system (Vibler), using Immobilon Western Chemiluminescent HRP Substrate (Merck, WBKLS0500). Western blot signals were quantified using ImageJ software. Antibodies are listed in the Table 2. Western blot membranes are presented together with molecular mass markers in the Additional file 1.
Radaszkiewicz T, & Bryja V. (2020). Protease associated domain of RNF43 is not necessary for the suppression of Wnt/β-catenin signaling in human cells. Cell Communication and Signaling : CCS, 18, 91.
Manipulation of cells (e.g., treatment conditions, genetic modifications)
dependent variables
Western blot signals quantified using ImageJ software
control variables
Cell lysis buffer composition (2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.002% bromphenol blue, 0.06 M Tris HCl, pH 6.8)
Protein electrophoresis and transfer onto PVDF membrane
Primary and secondary antibodies used for detection
controls
Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned
Annotations
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