Peter van Mourik

32 Chapter 2 at the penultimate base to enhance reaction specificity using established guidelines 6 . The optimal melting temperature of these primers was determined (62,5 ° C) by gradient Q-PCR experiments using cDNA from F508del/R117H and F508del/S1251N organoid cultures. PCR products of allele-specific R117H-CFTR reactions (R117H/ R117H organoids) were sequenced to verify the identity of the amplicons. Allele- specific RT-qPCR experiments were performed using similar reaction conditions as described above. Western blot analysis Organoids were lysed in Laemmli buffer (2% SDS, 10% glycerol and 63 mM Tris- HCl pH 6.8) supplemented with complete protease inhibitor (Roche, Ltd.). Protein lysates of each sample (50 μg) were used to perform gel electrophoresis, followed by protein transfer to an Immobilon-FL polyvinylidene fluoride (PVDF) membrane (Sigma-Aldrich). The membrane was blocked for 1 hour with 5% w/v milk powder (ELK) dissolved in Tris-buffered saline-Tween (TBST; 0.3% Tween, 10 mM Tris, pH 8, and 150 mM NaCl in H 2 O) and incubated o/n at 4°C with primary antibodies mouse α-CFTR 450, 570, 596 (Cystic Fibrosis Folding consortium); 1:5.000 and rabbit α-HSP90 (Braakman laboratory, Utrecht University; 1:50.000), followed by a 1 hour RT incubation with secondary antibodies IRDye 680RD donkey-α-rabbit IgG and IRDye 800CW donkey-α-mouse IgG (LI-COR, Inc.) diluted 1:20.000. All antibodies were diluted in 5% w/v ELK in TBST. Protein detection was performed on the LI-COR Odyssey 3 imaging system (LI-COR, Inc.) using default settings and image resolution. Densitometry analysis was performed using LI-COR Image Studio Lite 5.3 (LI-COR, Inc.). For CFTR protein expression, only mature CFTR protein (C- band) was quantitated. To achieve normalized values, C-band density was corrected for loading using the density of the corresponding HSP90 band and normalized to wild type CFTR protein expression. CFTR protein expression in the organoid cultures was measured in three independent experiments (n=3).