Suzanne de Bruijn

152 Chapter 3.2 RNA isolation and RT-qPCR Total RNA was isolated from treated cells using the Nucleospin RNA kit (Machery-Nagel) according to themanufacturer’s instructions. Subsequently, cDNAwas synthesizedusing the iScript cDNA synthesis kit (Bio-Rad) and 250 ng RNA as input material. Obtained cDNA was diluted twice (fibroblast cells) or five times (HEK293T cells) for quantitative PCR analysis. QPCR analysis was performed using GoTaq qPCR Master Mix (Promega) according to standardprocedures.Allele-specificprimersetswereemployedthatallowedtodistinguish themutant andwildtype RIPOR2 transcripts and included allele-specific forward primers (5’-GGAAGGAAACATCACAAAGAG-3’, mutant allele; 5’-AAGCAGCTGGTCAAGAGG-3’, wildtype allele), and a universal reverse primer (5’-GCAGCCTTCAGATTCTCC-3’). Relative expression levels as compared to the housekeeping gene GUSB (primers 5’-AGAGTGGTGCTGAGGATTGG-3’ and 5’- CCCTCATGCTCTAGCGTGTC-3’) were determined with the 2^ΔCt method. Western blot For protein isolation, cells were treated with a lysis buffer containing 50 mM Tris pH 7.5, 150 mM NaCl and 0.5% (v/v) Triton X-100. Protein lysates were supplemented with protein sample loading buffer (LI-COR) and DTT (final concentration 2 mM) and denatured at 70°C for 10 minutes. Proteins were separated on a 4-12% NuPAGE Bis-Tris gel (ThermoFisher) in NuPAGE MOPS SDS running buffer (ThermoFisher) and transferred overnight at 4°C in NuPAGE transfer buffer supplemented with 20% methanol to a nitrocellulose membrane (0.45 mm, Sigma-Aldrich). Membranes were blocked with 5% Blotto, non-fat dry milk, blocking buffer (Santa Cruz), and incubated with primary antibodies (1 hour, RT) and secondary antibodies (45 minutes, RT), in 0.5% blocking buffer. In between incubation steps, the membranes were washed three times for 10 minutes with PBS supplemented with 0.2% Tween-20. After a final wash with PBS, proteins were visualized using the Odyssey Infrared Imaging System (LI-COR). RIPOR2 protein was quantified using the Fiji software (version 1.47), and normalized against tubulin. Antibodies: anti-RIPOR2 (1:1000; cat.#17015-1-AP, Proteintech), anti-tubulin (1:2000; cat.# ab7291, Abcam), goat anti-rabbit Alexa Fluor 680 (1:20000; cat.#A21076, Molecular Probes) and goat anti-mouse IRDye800 (1:20000; cat.# 926-32210, LI-COR).