Sc 48794

Immunoblotting was performed as described (60 (link)). Briefly, cultured cells were lysed using radioimmunoprecipitation assay buffer supplemented with a protease inhibitor cocktail (Roche). Proteins were resolved with SDS–polyacrylamide gel electrophoresis, and after transfer to a nitrocellulose membrane were blocked in Odyssey blocking buffer (LI-COR). The following primary antibodies were used: human NAF1 (rabbit, ab157106, 1:1000; Abcam), mouse Naf1 (rabbit, Naf1 394, 1:250; Prosci), Myc (mouse, clone 4A6, 1:1000; Millipore), human dyskerin (rabbit, sc-48794, 1:250; Santa Cruz Biotechnology), and green fluorescent protein (mouse, 7.1 and 13.1, 1:1000; Roche) with loading controls actin (mouse, ab8226, 1:2000; Abcam), tubulin (rabbit, ab6046, 1:5000; Abcam), PARP (rabbit, 9542S, 1:1000; Cell Signaling Technology), or GAPDH (mouse, mAbcam9498, 1:1000; Abcam). Secondary antibodies were conjugated to dyes IR680 or IR800 (donkey, 1:10,000; LI-COR), and blots were visualized using an Odyssey scanner (LI-COR), with exception of the mouse Naf1 antibody that was visualized by horseradish peroxidase–linked antibody (rabbit, 7074, 1:10,000; Cell Signaling Technology) and enhanced chemiluminescent substrate (Thermo Scientific). Cell fractionation was performed using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific).

Total cellular lysates were subjected to SDS-PAGE, and protein was transferred to PVDF membranes using standard procedures. Detection of PARN and dyskerin was performed using primary antibodies to human PARN (Abcam, ab188333; 1:5,000 dilution) and dyskerin (Santa Cruz Biotechnology, sc-48794; 1:1,000 dilution) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (Bio-Rad, 170-5046; 1:15,000 dilution), followed by chemiluminescent detection using Clarity Western ECL substrate (Bio-Rad). Protein loading was determined using HRP-conjugated antibody to actin (Santa Cruz Biotechnology, sc-1615; 1:1,000 dilution) on the same membranes and used for normalization. Imaging and quantification of chemiluminescent signals was performed using the Bio-Rad ChemiDoc Touch imaging system. Graphing and statistical analyses were performed using GraphPad Prism.