Amersham protran 0.45 μm nitrocellulose membranes

For the dot blot analysis, 0.5, 1 and 2 µg of each sample were carefully pipetted on distinct spots of a nitrocellulose membrane (Amersham Protran 0.45 μm nitrocellulose membranes, Cytiva, Marlborough, MA, USA). For a negative control, the same amounts of bovine serum albumin (BSA) were also dotted in the membrane. After drying for 30 min at RT, the membrane was blocked for 2 h in 5% bovine serum albumin (BSA, Sigma-Aldrich) in TBS-T (20 mM Tris-HCl, 150 mM NaCl, pH 7.5 with 0.05% Tween20 (Sigma-Aldrich)). After washing in TBS-T, the membrane was incubated with the primary antibody (1:1000 in 1% BSA in TBS-T) overnight at 4 °C and then, with the secondary antibody (1:30,000 in 1% BSA in TBS-T) for 1 h at RT. The primary antibody used was the rabbit anti-collagen I antibody (1:1000, ab233639, Abcam plc, Cambridge, UK) and the secondary antibody was the goat anti-rabbit IgG, HRP-linked antibody (1:20,000, 7074, Cell Signaling Technology, Inc., Danvers, MA, USA). The blot was washed with TBS-T, incubated with the Clarity Western ECL substrate (Bio-Rad Laboratories) for 1 min and visualized using the Odyssey Fc Imaging System (LI-COR Biosciences, Lincoln, NE, USA).

HFF-WT cells were infected with HCMV-TB40-WT (MOI = 3), cells were harvested and lysed at 1 hpi (input) and every 24 hours until 96 hpi. SDS-PAGE was performed on Bio-Rad Mini-PROTEAN TGX 4–15% gels (Bio-Rad). Separated protein was blotted on Amersham Protran 0.45 μm nitrocellulose membranes (Cytiva). The membrane was cut, and the sections were subsequently stained with one of the primary antibodies against HCMV pp150 (kind gift by Eva-Maria Borst and Stipan Jonjic), anti-CMV ICP36 monoclonal antibody 10D8 (Virusys), and anti-IE1/2 (hybridoma supernatant [111 (link)], kind gift by Wolfram Brune) followed by a secondary antibody Goat Anti-Mouse IgG StarBright Blue 700 (Bio-Rad). An Rhodamine coupled Anti-GAPDH hFAB antibody (Bio-Rad) was used as loading control. The stained blots were imaged using a ChemiDoc MP imager (Bio-Rad).