Rabbit anti dyskerin antibody

In order to differentiate between endogenous dyskerin and exogenous overexpressed protein, immunofluorescent staining of dyskerin was performed, allowing examination of their respective location within ISK cell. Rabbit anti-dyskerin antibody (Santa Cruz Biotechnology, Dallas, TX, USA, 1:200) was added to the fixed cells, which were seeded onto coverslips in a 6 well plate. The secondary antibody was Alexa Fluor Anti-rabbit IgG (H + L), (Alexa Fluor 488 Conjugate), (Cell Signalling Technology, London, UK, 1:1000). The cells were mounted in DAPI containing medium (Vector Laboratories, Peterborough, UK,). Fluorescence was visualised with a Nikon Eclipse 50i microscope using NIS elements F software (Nikon, Tokyo, Japan). Rabbit and mouse isotype control antibodies were used as negative controls. Antibody details are provided in Table S3.

Lysates or elution from the oligo purification (OE) from wild-type or mutant telomerase samples were resolved on a 4-12% Bis-Tris NuPAGE gel (Invitrogen, Cat# NP0321BOX) then transferred onto a nitrocellulose membrane. Membranes were blocked for 1 hour at room temperature with 5% non-fat milk, prepared in phosphate buffer saline (PBS) supplemented with 0.2% Tween-20 (PBST), followed by an overnight incubation at 4 °C with primary antibody (1:2,000 rabbit anti-dyskerin antibody, Santa Cruz Biotechnology, Cat# sc-48794, lot E0214, RRID: AB_2091314 or 1:20,000 mouse anti-alpha tubulin, ProteinTech, Cat# 66031-1-Ig, lot 10004185). The following day membranes were washed 3 times with PBST, incubated with a secondary antibody (1:10,000 goat anti-rabbit Alexa-Fluor 680, Abcam Cat# ab175773, lot GR222353-8, or 1:10,000 goat anti-mouse Alexa-Fluor 680, Abcam Cat# ab175775, lot GR3273649-2) for 1 hour at room temperature and then washed 3 additional times with PBST before imaging on a Li-COR Odyssey imager. Densitometry for quantification was performed in ImageJ.