Pngase f p0704s

PrP^C-specific antibodies 3F4 and 8H4 were obtained from Signet laboratories (Dedham, MA, USA). 3F4 binds to an epitope on amino acids 109–112 (Human PrP sequence), while 8H4 binds an epitope in the C-terminal amino acids 145–180 of human and mouse PrP^{62 (link)}. Other primary antibodies were obtained from the following sources: anti-ferritin (F5012, Sigma Aldrich, USA), anti-DMT-1 (ab55812, Abcam, USA), anti-TfR (ab84036, Abcam, USA), anti-Tf (GTX21223, GeneTex, USA), and anti-β-actin (MAB1501, Millipore, USA) Secondary antibodies conjugated with HRP were from GE Healthcare (anti-mouse, LNA931V, anti-rabbit, LNA934V). Alexa fluor-conjugated secondary antibodies were from molecular probes, ImmPACT DAB (SK4105) from Vector labs, USA, PNGase F (P0704S) from NEB, USA and Lipofectamine 3000 transfection reagent from Invitrogen, USA. Radiolabeled ⁵⁹FeCl₃ was obtained from Perkin-Elmer, USA. siRNA against PrP (sc36318) and scrambled siRNA (sc37007) were purchased from Santa Cruz Biotechnology Inc, USA. For immunohistochemistry of hamster retinal sections PrP-6C2 (CVI-WUR, Lelystad, Netherland), ferritin (Jackson ImmunoResearch, West Grove, PA, USA), and Iba1 (019–19741, Wako Chemicals, USA) antibodies were used. FAC (F5879) and all other chemicals were from Sigma Aldrich, USA.

To remove N-glycans attached during heterologous expression in P. pastoris, 5 ml of purified recombinant Bgl3A was incubated with 5000 U (10 µl) of peptide-N-glycosidase F (PNGase F, P0704S, New England Biolabs, Ipswich, MA) at 37 °C for 12 h according to the manufacturer’s instructions. To remove O-glycans, 4 ml of PNGase F-treated Bgl3A was incubated with 10 µl of α-mannosidase (M7257, Sigma-Aldrich) for 24 h, followed by purification through anion exchange chromatography. Both N- and N-/O-deglycosylated enzyme samples were analyzed by SDS–PAGE.