Mini trans blot cell apparatus

Western blot was performed using total proteins and nuclear extracts obtained from approximately 2.0 × 10⁶ cells. Cells were collected following standard procedures. Then, 1.0 mL of ice cold RIPA Lysis buffer (Thermo Scientific, Waltham, MA, USA) was added, with freshly added Protease and Phosphatase Inhibitors Cocktails (Thermo Scientific). SOX2, WWTR1/TAZ and YAP1 were investigated in nuclear fractions, obtained using CelLytic™ NuCLEAR™ EXTRACTION KIT (Sigma-Aldrich). Total proteins and nuclear fractions were measured in the extract by the Bradford assay.
The nuclear fractions and the whole cell lysates were examined on Mini-PROTEAN TGX Gels 4–20% (Bio-Rad). Proteins were transferred from gels on Immun-Blot PVDF Membranes (Bio-Rad) in the transfer buffer (glycine, tris [pH 8.4] and methanol) using Mini Trans-Blot Cell apparatus (Bio-Rad). Membranes containing proteins were blocked with milk 5X (Sigma-Aldrich) in TBST and, subsequently, probed with primary and horseradish peroxidase conjugated secondary antibodies following standard procedures. Proteins transferred membranes were washed with TBST and developed with Pierce ECL Western Blotting Substrate (Thermo Scientific). Details are provided in the online Supporting Information.

Denatured proteins were first resolved in 4–12% Bis-Tris or 3–8% Tris-Acetate pre-cast gel in 1X NuPAGE SDS running buffer (MES, MOPS, or Tris-Acetate) for ~1.5 h at 150V and then (electro)transferred onto a nitrocellulose membrane for 1 h in a 1X Tris-glycine transfer buffer with 20% ethanol (120V, ~1.5 h) in a Bio-Rad Mini Trans-Blot Cell apparatus. After protein transfer, membranes were blocked with gentle agitation in 10% skimmed milk (MSK) solution in TBST for 1 h. Target proteins in the membrane were probed either individually or simultaneously using an optimized antibody cocktail prepared in 5% MSK/TBST solution; primary antibody cocktail (overnight incubation at 4°C): α-GFP (1:3000), α-mCherry (1:6000), α-Tdh3 (1:5000), α-actin (1:5000) and secondary antibody cocktail (1 h incubation at room temperature): goat α-mouse poly-HRP (1:15000), goat α-rabbit poly-HRP (1:10000), α-HA-HRP (1:15000), α-tubulin-HRP (1:2500). After extensive washing with TBST, membranes were added with chemiluminescent substrate (SuperSignal West Dura Extended Duration Substrate) to detect the immunoreactive bands using the Azure 280 or ImageQuant LAS 500 detection system. Signal quantification on TIFF files were made using Image Lab (Bio-Rad).

Northern blot analyses were performed with a DIG Northern Starter Kit (Roche), as previously described by Beckmann et al.36 (link). Briefly, total RNA (20 μg/sample) was denatured at 70 °C for 5 min, run on a 10% polyacrylamide–7 M urea gel, and then transferred to Hybond N⁺ membrane (GE Healthcare) with a Mini Trans-Blot Cell apparatus (Bio-Rad Laboratories). The membrane was prehybridized in ULTRAhyb® Ultrasensitive Hybridization Buffer (Ambion) for 45 min, and 3′-end DIG-labeled RNA probes were added. The membranes were then hybridized overnight at 64 °C in DIG Easy Hyb, according to the manufacturer’s protocol.