Ab116027

Protein preparations for Western blotting from transfected HEK293S cells and tissues are described in the Supplemental Information. Protein samples (1–100 µg, Table S6) were loaded alongside the Benchmark (Cat# 10748010, Life Technologies, Carlsbad, CA, USA), PageRuler (Cat# 26616, Thermofisher, Waltham, MA, USA) or ab116027 (Abcam, Cambridge, UK) protein ladders onto 4–12% SurePage Bis-Tris gels (GenScript, Piscataway, NJ, USA). Western blotting was performed as previously described [43 (link)]. Under some conditions, 2-(N-morpholino)ethanesulfonic acid (MES) running buffer was used to aid in the separation of lower molecular weight bands. Figure legends provide detailed information about the conditions used.

The cells of 2D cultures and 3D MCSs were harvested as pellets by centrifugation and washed with PBS. The whole-cell lysates were prepared by lysing pellets in RIPA buffer (150 mM NaCl, 50 mM Tris–HCL pH 7.4 (4 °C), 1% Tween-20 in TritonX, 1% sodium-deoxycholate, 10% SDS) containing protease inhibitor cocktail (Roche, Basel, Switzerland). Protein concentrations were evaluated with Pierce™ BCA Protein Assay Kit. The extracted proteins (20 µg) were mixed with loading dye and separated on SDS-PAGE. After that, they were transferred to a polyvinylidene difluoride membrane (Whatman, Dassel, Germany) under 250 mA, free voltage for 1.40 h. When finished transferring, the membranes were cut according to the prestained protein ladder (ab116027, Abcam, UK ) to cover the target band. The pre-cut membranes were blocked with 5% skim milk and probed with primary antibodies at 4 °C overnight. On the next day, the membranes were washed and probed with horseradish-peroxidase-conjugated secondary antibodies at room temperature for 1 h. Signals were detected using chemiluminescence ECL (Amersham, Italy). The list of primary and secondary antibodies used in this experiment were listed in supplementary Table S2.