Trans blot turbo pvdf nitrocellulose membrane

For each condition, 5 × 10⁶ BMDCs transfected at day 6 were lysed 8 h post transfection at 4 °C in 25 mM Tris pH 7.5, 50 mM NaCl, 0.1% NP40 and a protease inhibitor cocktail (Roche) during 1 h. Lysates were incubated with 20 μl of GFP-trap beads (Chromotek) for 5 h at 4 °C under rotation, washed four times in 1 ml lysis buffer, dried and resuspended in Laemmli Buffer, loaded on a 4–20% TGX gradient gel (Bio-Rad) and transferred on a Trans-Blot Turbo PVDF/Nitrocellulose membrane (Bio-Rad). Membranes were blocked with PBS+0.05 %Tween20+5% BSA, incubated with primary antibodies (1/1,000 anti-myosin IIA HC and 1/10 homemade anti-human Ii C-term rabbit serum), washed and incubated with secondary antibodies (1/5,000 HRP-conjugated antibodies). Signals were revealed with the SuperSignal West Dura substrate (Thermo Scientific).

Immunoblotting was performed as previously described (Vargas et al., 2016 (link)). Briefly, DCs were lysed for 2 min in a buffer containing 100 mM Tris, 150 mM NaCl, 0.5% NP-40 and a protease inhibitor cocktail tablet (Roche). Fifty micrograms of soluble extracts were loaded onto a 4–20% TGX gradient gel (BioRad) and transferred onto a Trans-Blot Turbo PVDF/Nitrocellulose membrane (BioRad). The membrane was blocked, incubated sequentially with the appropriate antibodies and revealed using the SuperSignal West Dura substrate (Thermo Scientific).