Anti streptavidin

Lysates and eluates were run on 4–12% polyacrylamide gels and transferred to PVDF (Immobilon-P, EMD Millipore, Burlington, MA, USA) for 1.5 h at 100 V constant voltage. Blots were blocked for 30 min with PBST (phosphate buffer saline, 0.05% Tween, Thermo Fisher Scientific) 5% dry non-fat milk (w/v) and immunoblotted with appropriate antibodies. All antibodies were diluted in PBST 2% milk (w/v). Primary antibodies were incubated overnight at 4 °C, while secondary antibodies were incubated for 1 h at RT. Antibodies used were anti-FLAG (Sigma-Aldrich, St. Louis, MO, USA, 1:5000 dilution), anti-Streptavidin (Thermo Fisher Scientific, 1:1000 dilution), anti-HA (1:5000, Thermo Fisher Scientific) anti-GAPDH (Thermo Fisher Scientific 1:10,000 dilution), anti-PCM1 (Santa Cruz Biotechnology, Dallas, TX, USA, 1:500 dilution), anti-PIBF1 (Santa Cruz Biotechnology, 1:500 dilution), anti-NudC (Santa Cruz Biotechnology, 1:500 dilution), anti-GT335 (Adipogen, San Diego, CA, USA, 1:5000 dilution), goat anti-rabbit HRP (Bio-Rad, Hercules, CA, USA, 1:5000 dilution) and goat anti-mouse HRP (Bio-Rad, 1:5000 dilution). Western blots were visualized with Luminata Forte (Merck Millipore, Darmstadt, Germany) and a VersaDoc imaging system (Bio-Rad). Image intensity histograms were adjusted, and images were analyzed with ImageLab software (version 6.1.0 build 7, Bio-Rad).

HeLa cells were grown on sterilized coverslips until 60%–80% confluent and then transfected using Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA) for 24 h. Cells were washed twice with ice-cold PBS and then fixed with 4% (w/v) paraformaldehyde in PBS for 10 min, and permeabilized with acetone and methanol (1:1) for 30 s. Cells were then incubated with primary antibodies diluted in PBS with 10% (v/v) normal goat serum for 1 h and with the secondary antibody under the same conditions. The primary antibody was anti-streptavidin (Thermo, Waltham, MA, USA) that marked by DyLight 488 (Molecular Probes, KPL, Gaithersburg, MD, USA). Hoechst 33258 (Sigma, St. Louis, MO, USA) was used to stain the nuclei. Fluorescence on the processed slips was analyzed using a fluorescence microscope (Zeiss, Oberkochen, Germany).