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 .
Antibodies
| Antibody | Manufacturer | Dilution and application | Reference |
|---|---|---|---|
| LRP6 (C47E12) | cs-3395, Cell Signaling Technology | 1:1000, WB | [28 ] |
| LRP6 (Phospho-S1490) | cs-2568, Cell Signaling Technology | 1:1000, WB | [28 ] |
| β-actin | CS-4970, Cell Signaling Technology | 1:3000, WB | [26 (link)] |
| DVL-2 | CS-3216, Cell Signaling Technology | 1:1000, WB | [26 (link)] |
| DVL-3 | CS-3218, Cell Signaling Technology | 1:1000, WB | [26 (link)] |
| HA-11 | MMS-101R, Covance | 1:2000 WB; 1:500, IF | [28 ] |
| a-mouse IgG HRP | A4416 | 1:4000 WB | – |
| a-rabbit IgG HRP | A0545 | 1:4000 WB | – |
| a-mouse Alexa Fluor™568 | A10037, Thermo Fisher Scientific | 1:600 IF | – |
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