Versadoc 4000 mp imager

RK13 cells were (co-)transfected with plasmids expressing the indicated gH variants with or without the gL-expression plasmid using Lipofectamine 2000 (Thermo Fisher Scientific), and pEGFP-N1 (Clontech) served as negative control. For virus characterization, RK13 cells were infected with the different mutants and PrV-Ka at a MOI of 3. Transfected and infected cells were harvested one day later, washed with phosphate-buffered saline (PBS), and lyzed in sample buffer (0.13M Tris-HCl, pH 6.8; 4% SDS; 20% glycerol; 0.01% bromophenol blue; 10% 2-mercaptoethanol). After boiling for 3 min, proteins were separated on SDS-10% or 12% polyacrylamide gels and transferred to nitrocellulose. Membranes were blocked with 5% skimmed milk in tris-buffered saline with 0.1% Tween-20 (TBS-T), and probed with monospecific PrV antisera diluted in TBS-T (anti-gH 1:15.000, anti-gL 1:1.000, anti-UL38 1:100.000) [7 (link),9 (link)] or an anti-tubulin monoclonal antibody (Sigma-Aldrich, Taufkirchen, Germany) as loading control. Bound antibody was detected after incubation with horseradish peroxidase-conjugated α-rabbit or α-mouse IgG (Invitrogen, Waltham, MA, USA) using the Clarity Western ECL Substrate (BioRad). Signals were recorded with a Versa DOC 4000 MP imager (BioRad, Feldkirchen, Germany).

RK13 cells were harvested 24 h after transfection with 600 ng of the expression plasmids encoding wild-type gB or the different N-glycosylation mutants using lipofectamine 2000 (Thermo Fisher Scientific, Darmstadt, Germany). Cells were lysed, and protein samples were separated by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to nitrocellulose membranes. Membranes were incubated with monoclonal PrV gB antibodies (c15-b1 or A20-c26 at 1:10 dilution) [18 (link),47 (link)] and peroxidase-conjugated secondary antibody (Dianova, Hamburg, Germany). Clarity Western ECL substrate (Bio-Rad Laboratories, Feldkirchen, Germany) and a VersaDoc 4000 MP imager (Bio-Rad Laboratories, Feldkirchen, Germany) were used for detection.

Infected or transfected cells were harvested after 18 h and lysed, and protein samples were separated by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred to a nitrocellulose membrane. Membranes were incubated with appropriate antibodies. Peroxidase-conjugated secondary antibody (Jackson ImmunoResearch) was detected with Clarity Western ECL substrate (Bio-Rad) and recorded with a VersaDoc 4000 MP imager (Bio-Rad) using the Quantity One software (version 4.6.9).

Lysates of parental, single and double knockout RK13 as well as of Vero and HeLa cells were separated in sodium dodecyl sulfate (SDS)-10% polyacrylamide gels. Proteins were transferred to nitrocellulose membranes and blocked with 6% skimmed milk. Parallel blots were incubated with the polyclonal rabbit sera specific for VAPA (1:2.500; Invitrogen # PA5-22188; raised against a synthetic peptide corresponding to a region within amino acids 29 and 122 of human VAPA ID#Q9P0L0; ThermoFisherScientific), VAPB (1:5.000; Invitrogen # PA5-53023; corresponding to amino acids 105-216 of human VAPB ID#O95292; ThermoFisherScientific), or with a monoclonal α-tubulin antibody (1:10.000; Sigma, Taufkirchen, Germany). Bound antibodies were detected after incubation with peroxidase-conjugated anti-rabbit or anti-mouse antibodies (Dianova, Hamburg, Germany) using the Clarity Western ECL substrate (Bio-Rad Laboratories, Feldkirchen, Germany). Signals were recorded with a VersaDoc 4000 MP imager (Bio-Rad Laboratories) using the Quantity One software (version 4.6.9).