Hpa018019

Frozen sections were prepared by fixing tissues overnight in 4 % paraformaldehyde, followed by cryoprotection in 30 % sucrose at 4 °C for two days and sectioning with given thickness for histological and immunofluorescence analysis. For morphological analysis, Section (5 μm) were stained with hematoxylin and eosin to have images acquired with a Leica DMRXA2 microscope. For immunofluorescence analysis, Section (6–8 μm) were treated following the standard protocol with following antibodies: rabbit anti-OTG1 (Sigma HPA018019, 1:1000), Alexa 488 conjugated rabbit-anti-Giantin (Covance A488-114L, 1:1000), rabbit anti-insulin (Santa Cruz sc-9168, 1:1000), goat anti-glucagon (Santa Cruz sc-7780, 1:1000), donkey anti-goat IgG-FITC (Chemicon AP180F, 1:2000), donkey anti-rabbit IgG-Cy3 (Millipore AP182C, 1:2000).

Western blot of CCDC186 protein was performed in muscle biopsy of P1.1 and in fibroblasts of P1.2, as well as control tissues. Material from P2.1 was not available. Muscle extracts were prepared in tissue extraction buffer (250 mM mannitol, 75 mM sucrose, 10 nM Tris-HCl, 0.1 mM EDTA) and centrifuged at 600 g at 4 °C for 20 min. Fibroblasts were lysed using RIPA buffer containing a protease inhibitor cocktail (1862209, Merck, Darmstadt, Germany). Briefly, cells were scraped in RIPA lysis buffer, incubated on ice for 10 min, and centrifuged at 10,000 g at 4 °C for 10 min. Protein extracts (40 ug) were subjected to SDS-PAGE, electroblotted, and visualized by immunostaining with specific antibodies followed by colorimetric detection (Opti-4CNTM Substrate Kit, Bio-Rad, Hercules, CA, USA). The antibodies used in this study were: anti-CCDC186 (HPA018019, Sigma Aldrich, Burlington, MA, USA) and anti-beta actin (ab115777, Abcam, UK). Membrane images were quantified using ImageJ software 1.53t.

MCF-7 cells were plated in T25 flask at a density of 2 × 10⁶ cells/flask. At about 70% confluence, the culture medium was removed, and cells incubated with fresh complete medium for 48 h. For abrogation of the effect of c10orf1118 protein, CM from MCF-7 cells was harvested, centrifuged to remove cell debris and incubated with gentle agitation for 1h at 37 °C with 4 µg/mL (final concentration) of anti-c10orf118 rabbit polyclonal antibody (HPA018019; Sigma). The same concentrations of α-actin (sc-1616; Santa Cruz) were used as control. NHDF cells were plated in 6-well at a density of 5 × 10⁵ cells/well. Then, 800 µL of pre-incubated CM with blocking antibodies were added. After 48h, NHDF cells were harvested for RNA in TRI Reagent^® to study HASes gene expression.