Immuno-blotting of protein samples

Protein concentration was measured using a Direct Detect Infrared Spectrometer (Merck). 20 µg of proteins were separated on a NuPAGE 4–12% Bis-Tris gel (Thermo Fisher Scientific). Proteins were transferred with an iBLot2 device (Invitrogen) on a nitrocellulose membrane and blocked for 1 hr in 1x Licor TBS Blocking Buffer (Licor). Primary antibodies were incubated over night at 4°C. Licor secondary antibodies were incubated for 45 min at room temperature (RT) and images acquired with an Odyssey CLx scanner (Licor). The following antibodies were used: anti-HA (ab9110), anti-FLAG (Sigma #F1804), anti-GFP (ab13970), anti-Piwi (described in Brennecke et al., 2007 (link)), anti-Nxt1 (Herold et al., 2001 (link)), anti-Histone H3 (ab10799), anti-Tubulin (ab18251), mouse anti-Panx (Sienski et al., 2015 (link)), IRDye 680RD Donkey anti-Rabbit IgG (H + L) (Licor), IRDye 800CW Donkey anti-Mouse IgG (H + L) (Licor), IRDye 800CW Goat anti-Rat IgG (H + L) (Licor).