Pierce precise protein gels

For western blot analysis, HEp-2 cells were infected with VACV_NYCBOH at an MOI of 0.2 and cultivated for 3 to 4 days. Cells were lysed using RIPA lysis buffer supplemented with the HALT Protease Inhibitor cocktail (Thermo Fisher Scientific) and protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific). A protein lysate from mock-infected HEp-2 cells was used as the negative control. Proteins were separated on 8 to 16% precast gradient PAA gels (Pierce Precise™ Protein Gels, Thermo Fisher Scientific) and blotted onto a 0.2 μm PVDF membrane (VWR, Darmstadt, Germany). A PageRuler™ pre-stained standard (Fermentas, St. Leon-Rot, Germany) was used as the molecular weight marker. Membranes were blocked in TBS-T supplemented with 5% skim milk (Carl Roth) at 4°C overnight. Primary antibodies were incubated for 1 h at RT diluted in TBS-T supplemented with 1% skimmed milk, as were HRP-labelled species-specific secondary antibodies. Detection was done via chemiluminescence after 5 min incubation with ECL western blotting substrate (Thermo Fisher Scientific).