Jos Jansen

2 33 CCDC115 Deficiency Causes a Disorder of Golgi Homeostasis with Abnormal Protein Glycosylation software v.1.46 (see Web Resources). Primary antibodies used are as follows: anti-V5-Tag (1:200, Life Technologies, #R960-25), anti-beta COP (1:1,000, Abcam, #ab2899), anti-SEC31A (1:500, Sigma-Aldrich, #HPA005457), anti-Giantin (1:1,000, BioLegend, #prb-114c), anti-ERGIC53/p58 (1:200, Sigma-Aldrich, #E1031), anti- PDI (1:500, Abcam, #ab3672), anti-Calnexin (1:200, StressMarq Biosciences, #SPC-108). Secondary antibodies are as follows: Alexa Fluor 488-conjugated goat antimouse IgG (H+L) (1:1,000, Life Technologies, #A-11029) and Alexa Fluor 568-conjugated goat anti-rabbit IgG (H+L) (1:1,000, Life Technologies, #A-11011). IEF of Tf and ApoC-III Tf IEFwas performed as previously described.(21) In short, 10ml serumor plasma sample was added to a solution containing iron and NaHCO3, electrophorized on a 5–7 pH gradient gel, and incubated with 60 μ l polyclonal rabbit anti-Tf antibody (Dako, #A0061). Quantification of the gel was done with Image Quant software (TotalLab). Neuraminidase treatment was performed when a Tf polymorphism was suspected as described earlier.(21) ApoC-III IEF was performed as described, but with small modifications. (13) In short, 2 μ l of serum/plasma was 15x diluted with saline solution. Before electrophoresis, the gel was rehydrated in a solution containing 8 M urea. After blotting on a nitrocellulose membrane filter, the blot was washed and blocked before incubation with anti-ApoC-III (1:2,000, Rockland, #600-101-114). After incubation with the secondary anti-goat-HRP antibody (1:5,000, Thermo Scientific, #31402) and ECL reagent (Pierce), the blot was visualized on a LAS3000 imaging system (Fujifilm). Blot quantification was done with Image Quant software. MALDI-LTQ Mass Spectrometry Mass spectrometry of total plasma N-glycans was performed as described earlier.(22) To summarize, glycans from 10 μ l plasma were cleaved with PNGaseF (NE Biolabs) and incubated overnight. After purification on graphitized carbon SPE columns, the glycans were permethylated, purified again, and eluted in 50 μ l of 75% v/v aqueous acetonitrile. The glycans were dried and resuspended in a methanol/sodium acetate mixture for spotting. Measurements were done on a vMALDI-LTQ (Thermo Scientific).