Mouse anti calnexin

The sEVs were lysed in a reagent containing a cocktail of protease inhibitors to extract proteins. These proteins were subjected to Western blotting assay using standard procedures. Briefly, after resolution by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), proteins were electrotransferred to a cellulose nitrate membrane (Millipore), which was subsequently incubated at 4°C overnight with either mouse anti-CD63 (1:2000, Abcam), mouse anti-CD9, mouse anti-Tsg101, mouse anti-Alix (all diluted to 1:2,000, Abcam), or mouse anti-Calnexin (1:3,000, Abcam). The complex was then incubated with IRDye 680 secondary antibody, anti-rabbit, or anti-mouse for 1 h at normal laboratory temperature and then characterized by Odyssey infrared imaging system (LI-COR Biosciences).

Sperm samples were stained as previously described^{9 (link)}. Antibodies were diluted 1:400 (SpSLC9C1-SU1) and 1:750 (SpSLC9C1-SU2) in 0.1 M phosphate buffer (pH 7.4) in the presence of 0.5% Triton X-100 and 5% chemiblocker (Millipore). Primary antibodies were incubated overnight at 4 °C and visualized by goat-anti-rabbit A488 (1:500, 20 min incubation at RT, ThermoFisher Scientific A-11034, Rockford, USA). In CHO cells, SpSLC9C1-HA was probed by a rat-anti-HA antibody (1:1000, 1 h incubation at RT, Roche Applied Science) and visualized by goat-anti-rat A488 (1:400, 20 min incubation at RT, ThermoFisher Scientific A-11006). Membrane sheet preparations were obtained by sonification (0.1 s, 5%, Vibracell, Sonics & Materials, Newtown, USA) of CHO cells plated on poly-lysine (0.1 mg ml⁻¹, Sigma Aldrich) coated glass coverslips (Marienfeld-Superior, Lauda-Königshofen, Germany) in HEPES-buffered solution (1 mM DTT, 0.2% mPIC v/v). As marker for outer plasma membrane, CHO cells were additionally transfected with membrane-bound CAAX-RFP protein. For labeling the ER, a calnexin antibody was used (mouse-anti-calnexin, 1:500, Abcam ab31290, Cambridge, UK).

The following antibodies were used in this study: Mouse anti-β-Actin (Western blotting [WB] 1:5000, Sigma-Aldrich; A5316), mouse anti-FYCO1 (immunofluorescence [IF] 1:300, Abnova; H00079443-A01), rabbit anti-FYCO1 (IF 1:200, Invitrogen; PA5-45,805), mouse anti-GAPDH (WB 1:3000, Abcam; ab9484), rabbit anti-LAMP1 (IF 1:300, Merck Life Science; L1418), goat anti-mCherry (IF 1:200, OriGene; AB0040-200), rabbit anti-PARP (WB 1:1000, Bionordika; B9542S), rabbit anti-Protrudin (WB 1:7500, Protein Tech Group, 12680-1-AP), rabbit anti-RAB7 (IF 1:50–100, Cell Signalling Technology; D95F2), mouse anti-VAMP7 (IF 1:300, Synaptic Systems; 232 011), mouse anti-Vinculin (WB 1: 3000, Sigma; V9131), mouse-anti-Calnexin (IF 1:200, Abcam, ab22595), mouse anti-myc (IF 1:10, 9E10), and mouse-anti-GFP (IF 1:400, Merck Life Science 11814460001). The secondary antibodies were obtained from Jackson ImmunoResearch, Molecular Probes and LI-COR.