Magic mark western standard

Western blot was done by running samples in 4-12% of NuPAGE Bis-Tris Mini Gels (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s protocol and blotted onto a 0.45 μm PVDF membrane (Millipore, Billerica, MA, USA). Membrane blocking was performed by using TBS-T (TBS with 0.1% Tween-20; Sigma Aldrich) containing 5% (w/v) dried skimmed milk for 1 hour. The protein was probed by using an appropriate primary antibody overnight at 4°C. After washing the membrane 3 times with TBS-T, an appropriate secondary antibody was added for 1 hour at room temperature. After 2 washes with TBS-T and 2 washings with washing buffer, antigen-antibody complex was visualized by using SuperSignal™ West Pico Chemiluminescent Substrate (Cat.#34080 Thermo Fisher Scientific, Rockford, IL, USA). Magic-Mark™ Western standard from Invitrogen Life Technologies was used to estimate the molecular mass of the detected proteins.

Western blot was performed by separating protein samples on 4–12% NuPAGE Bis-Tris Mini Gels (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s protocol. Proteins were blotted onto a 0.45 μm PVDF membrane (Millipore, Billerica, MA, USA), and blocking was performed using TBST (TBS with 0.1% Tween-20; Sigma-Aldrich) containing 5% (w/v) dried skimmed milk for 1 h. The protein was probed by incubating the membrane with the primary antibody overnight at 4 °C. After washing the membrane 3 times with TBST, an appropriate secondary antibody was added for 1 h at room temperature. After 2 washes with TBST and 2 washes with washing buffer, antigen-antibody complex was visualized using SuperSignal™ West Pico Chemiluminescent Substrate (cat.no. 34080 Thermo Fisher Scientific, Rockford, IL, USA). The Magic-Mark™ Western standard from Invitrogen Life Technologies was used to estimate the molecular mass of the detected proteins. For the detection of MCPyV LTag, CM2B4 (Santa Cruz Biotechnology, Dallas, TX, USA; cat. no. sc-136172). ERK2 and GADPH antibodies were from Santa Cruz (cat. no. sc-154 and sc-47724, respectively).

Samples were analysed by 10-20% SDS-PAGE Bis-Tris glycine ClearPage gels (Invitrogen Life Technologies) according to the manufacturer’s protocol and blotted onto a 0.45 μm PVDF membrane (Millipore, Billerica, MA, USA). Immunoblotting was performed by first blocking the membrane with PBS-T (PBS with 0.1% Tween-20; Sigma Aldrich) containing 10% (w/v) dried skimmed milk for 1 hour and probed with the appropriate primary antibody overnight at 4°C. After 3 washes, the membrane was incubated with the appropriate secondary antibody for 1 hour at room temperature. After 4 washes, antigen-antibody complexes were visualized using CDP Star (Tropix, Bedford, MA, USA) substrate and Lumi-Imager F1 from Roche (Basel, Switzerland). Magic-Mark™ Western standard from Invitrogen Life Technologies was used to estimate the molecular mass of the detected proteins.