Anti-HA (rabbit polyclonal)

10 µg of total protein extracted freshly from mutant HEK293 cells with RIPA buffer and denatured at 65°C for 5 min were separated on  15% SDS-PAGE  and blotted overnight to PVDF (pore size of 0.4 um), using wet transfer. Blot was blocked with 5% milk or 2 h at RT and then incubated in the primary antibody at a dilution of 1: 5 000 in a rotator for 2.5 h in 5% milk. The membrane was then placed in a clean container and rinsed 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602) diluted to 1:20 000 in  for 1 h at RT with agitation. The blot was washed as above and developed with Agrisera ECLSuperBright. Exposure time was 30 seconds.

Courtesy Dr. Małgorzata Wessels, Department of Medical Biochemistry and Biophysics, Umeå University, Sweden

HEK293 cells were transfected with the indicated plasmid (SARS-CoV-2 N myc tagged, PMID: 34799561, TBEV NS3-HA, PMID: 29321318, Rab5 mcherry, Addgene: 4920, GFP-HIS) using genejuice transfection reagent (EMD Millipore) according to the manufacturer’s instructions. After 24 hours of transfection, cells were fixed in 4% formaldehyde and permeabilized in PBS containing 0.5% Triton X-100 and 20 mM glycine. Then, cells were stained with the primary anti-HA epitope tag, rabbit polyclonal antibodies at a concentration of 1 μg/mL for 1 hour at room temperature. Followed by three washes in PBS. Cells were then stained using secondary antibodies, donkey antirabbit Alexa488 (1:500, Thermo Fisher Scientific, a21206 in PBS containing 2% BSA for 1h at RT. Nuclei were stained using DAPI (1 μg/mL). Images were acquired using a Leica SP8 Laser Scanning Confocal Microscope with a 63x oil objective (Leica) and Leica Application Suit X software (LAS X, Leica). Anti-NS3 antibodies are used as a control.

Courtesy of Dr. Anna K Överby, Molecular Infection Medicine Sweden (MIMS),Section of Virology. Department of Clinical Microbiology Umeå University, Sweden