Supersignal west pico chemiluminescent substrate detection reagent

Roots of 7-days-old seedlings expressing NET4A::NET4A-GFP were homogenized in liquid nitrogen and solubilized in extraction buffer (200 mM Tris pH 6.8, 400 mM DTT, 8% SDS, 40% glycerol, 0.05% bromphenol blue). After incubation at 95 °C for 5–10 min, samples were centrifuged and the proteins contained in the supernatant separated using SDS-PAGE (10% gel). For blotting a polyvinylidene difluoride (PVDF) membrane (Immobilon-P, pore size 0.45 µm, Millipore, Burlington, MA, USA) was used and after blocking with 5% skim milk powder in TBST (150 mM NaCl, 10 mM Tris/HCl pH 8.0, 0.1% Tween 20), the membrane was probed with a 1:20,000 dilution of mouse anti-GFP antibody (JL-8, Roche, Roche Holding AG, Basel, Switzerland) or mouse anti-alpha-tubulin antibody (B-511, Sigma). As secondary antibody, a 1:20,000 dilution of horseradish-peroxidase-conjugated goat anti-mouse antibody (pAB, Dianova, Hamburg, Germany) was used. Signals were detected using the SuperSignal West Pico chemiluminescent substrate detection reagent (Thermo Scientific) and quantified using Fiji software. Signal intensities of GFP were normalized to alpha-tubulin and statistical evaluation was performed using Graphpad Prism 5 software.

Ovaries were dissected in Grace’s medium and processed for IP as described previously [16 (link)]. For western blotting, one-half ovary equivalent lysate was loaded into each lane of an 8% or 10% SDS-PAGE. The following primary antibodies were used: mouse anti-Aub (1:1,000, from Dr Siomi), mouse anti-Ago3 (1:500, from Dr Siomi), rat anti-Tap (1:1,000, this study), mouse anti-c-Myc 9E10 (1:5000, Sigma), mouse anti-HA (1:5,000, Roche, BASEL, Switzerland), mouse anti-FLAG M2 and its horseradish peroxidase (HRP)-conjugated secondary antibody (1:1,000, Sigma), guinea pig anti-Vas (1:5,000) [16 (link)], rabbit anti-Piwi (1:500, Abcam, Cambridge, England, United Kingdom Ab5207), mouse anti-Piwi (1:50, from Dr Siomi), rabbit anti-SpnE (1:500, from Dr Dahua Chen) and mouse anti-α-Tubulin DM1A (1:1,000, Santa Cruz Biotechnology, Santa Cruz Biotechnology, Dallas, Texas, U.S.A.). Immunoreactive bands were visualized using HRP-conjugated goat anti-guinea pig (Dako, Dako North America, Inc. Carpinteria, CA, USA), anti-rabbit, anti-rat or anti-mouse secondary antibodies (Bio-Rad, Hercules, CA, United States of America) at 1:5,000, and developed with the SuperSignal West Pico Chemiluminescent Substrate detection reagent (Thermo Scientific).

Cell pellets were lysed in 1 × sample buffer containing phosphatase and protease inhibitors. The lysates were sonicated, quantified and boiled for 10 min. Equal amount was electrophoresed in 7.5% or 4–20% SDS–polyacrylamide gels (Bio-Rad, Hercules, CA, USA). After transferring the proteins on to nitrocellulose membrane, filters were incubated in 5% non-fat dry milk in PBST (1X PBS plus 0.2% Tween-20) for 1 h. Blot was probed with p21 antibody (1:1000 dilution, Cell Signaling Technology, Danvers, MA) or TET2 antibody (EMD Millipore) in 1:2000 dilution and ß-actin antibody (1: 10,000 dilution) overnight at 4 °C in PBST, and subsequently incubated with a horseradish peroxidase-conjugated secondary antibody (BioRad) for 1 h at room temperature. Immuno-bound antibodies were detected by Super Signal West Pico Chemiluminescent Substrate detection reagent (Pierce/Thermo Scientific, Rockford, IL, USA) and visualized by autoradiography. The band density on the scanned images was quantified by ImageJ software (ImageJ.net).