?(Fig.2).2). in cells with trisomy 21. The impact of on the expression of (alpha chain component of FcRn) was investigated by knock down and overexpression of the APP protein. knock down increased the expression of mRNA by?~?60% in both diploid and trisomic cells. Overexpression of APP in diploid fibroblasts and HepG2 cells resulted in a decrease in and FcRn expression. Our results indicate that the intracellular traffic of IgG is altered in cells with trisomy 21. This study lays the foundation for future investigations into the role of FcRn in the context of DS. and knock down, cells were transfected using Dharmafect 4 transfection reagent (T-2004-02, Dharmacon) following the manufacturers recommendations. Briefly, 2 104 Diprotin A TFA cells per well were seeded into 24-well plates 24 h prior to transfection in antibiotic free media, and transfected with 5 nM siRNA. Cells were incubated at 37 C in 5% CO2 for a total of 96 h, with replacement of transfection medium 24 h post-transfection. Non-Targeting siRNA Control Pool (NS-siRNA, D-001206-13-05), siRNA Pool targeting (M-003731C00-0005), and siRNA against were obtained from Dharmacon. The siRNA against was designed using the web-based software OligoWalk (target sequence “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_001136019.2″,”term_id”:”226958651″,”term_text”:”NM_001136019.2″NM_001136019.2, Table S2)33. For expression of proteins with fluorescent tags, cells were transfected using ViaFect transfection reagent (E4981, Promega) following the manufacturers recommendations. Briefly, 5 104 cells per well were Diprotin A TFA seeded into 24-well plates 24 h prior to transfection in antibiotic free complete media, and transfected with 500 ng cDNA ORF Clone C-GFPSpark tag (HG10703-ACG, Sinobiological), or co-transfected with cDNA ORF Clone C-GFPSpark tag (HG11604-ACG, Sinobiological) and cDNA ORF Clone (HG11976-UT, Sinobiological). For control conditions, cells were transfected with an empty vector (PCMV6XL5, Origene). Cells were incubated at 37 C in 5% CO2 for a total of 48 h. Quantitative real-time polymerase chain reaction Total RNA was isolated from cells using Trizol reagent following the manufacturers instructions (Thermo Fisher). (alpha chain component of FcRn) and mRNA expression was analyzed with specific primers (Table S2). Total RNA (12.5 ng) was reverse transcribed and amplified with the iTaq Universal SYBR Green One-Step Kit (Bio-Rad). and the reference gene were amplified in parallel in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) with the following cycling parameters: 50 C for 10 min (reverse transcription), 95 C for 1 min, followed by 44 cycles of 95 C for 10 s, 60.5 C for 20 s. Calibration curves were prepared to analyze linearity and PCR efficiency. qRT-PCR data were analyzed with CFX manager Software (Bio-Rad). The Ct method was utilized for determining the relative abundance of and mRNA. Immunoblotting Cell lysates (20 g) were denatured with NuPAGE LDS sample buffer containing NuPAGE sample reducing agent and protease inhibitor cocktail (Thermo Fisher Scientific), and boiled at 70 C for 10 min prior to use. Proteins were separated by gel electrophoresis Diprotin A TFA using NuPAGE Novex 4C12% BisCTris precast gels and transferred onto PVDF membranes using the iBlot Gel Transfer Device (Thermo Fisher Scientific). Membranes were blocked with 5% non-fat milk in 0.2% Tween 20- phosphate-buffered saline (PBS) for IL7R antibody 1 h at room temperature and then probed with mouse monoclonal anti-FcRn antibody (1:100, Santa Cruz Biotechnology Cat# sc-271745, RRID:AB_10707665), mouse monoclonal anti-APP antibody (1:100, Thermo Fisher Scientific Cat# 13-0200, RRID:AB_2532993), rabbit monoclonal anti-APP antibody (1:100, Abcam Cat# ab133588, RRID:AB_2629851) or rabbit anti-Rab11 (1:250, Thermo Fisher Scientific Cat# 71-5300, RRID:AB_2533987) overnight at 4 C. Next, membranes were incubated with StarBright Blue 700 goat anti-mouse IgG secondary antibody (1:2500, Bio-Rad, Cat# 12004159, RRID: AB_2884948), StarBright Blue 520 goat anti-rabbit IgG secondary antibody (1:2500, Bio-Rad, Cat# 12005870, RRID: AB_2884949) and hFAB rhodamine anti-tubulin antibody (1:2500, Bio-Rad, Cat# 12004165, RRID: AB_2884950) for 1 h at room temperature. Immunoreactive bands were visualized in a ChemiDoc MP gel imaging system (Bio-Rad). Densitometric analysis was performed Diprotin A TFA using Fiji (ImageJ) software31. Data processing and statistical analysis Data processing was performed with Excel 2016 (Microsoft Office). Statistical analyses were performed with GraphPad Prism version 8. The D’Agostino & Pearson omnibus normality test was used to determine the normality of data sets. Comparisons between the means of two groups were performed with the Students t-test or MannCWhitneys U test for sets with normal and non-normal distributions, respectively. Spearmans rank-order test was used Diprotin A TFA for correlation analyses. Results Intracellular transport of IgG.