Advanced fluorescence imager

Cells were washed twice with ice-cold D-PBS before lysis buffer (10 mM Tris/HCl (pH 7,5), 150 mM NaCl, 0.5 mM EDTA, 1% NP-40 including protease inhibitor) was added to the apical chamber of the filter insert and incubated for at least 30 min at 4 °C. Cells lysates were centrifuged at 15,000 × g and 4 °C for 10 min and supernatants were stored at −20 °C until further processing. Fifty micrograms of each sample was separated by reducing SDS-PAGE under denaturing conditions and transferred onto nitrocellulose membrane by semi-dry western blotting. Detection of ACE2 was performed by incubation with anti-hACE2 antibody (R&D Systems (AF933); 1:500) and suitable secondary antibody coupled to horseradish-peroxidase (HRP) (1:10,000; Agilent Technologies, Santa Clara, USA). Equal loading of samples was controlled with immunostaining of actin. SuperSignal™WestDura Extended Duration Substrate was added to the membrane and the resulting chemiluminescence was detected using an Advanced Fluorescence Imager (Intas). Uncropped Western Blot membranes are depicted in Supplemental Fig. 9.

Caco-2 cells were seeded in 12-well plates in confluent monolayers and pre-treated with BAY598 at the indicated concentrations for 24 h. The cells were infected with a 1:10,000 dilution of the recSARS-CoV-2_d6-YFP reporter virus (d6-YFP) [37 (link)]. After 2 h, the inoculum was removed and the cell culture medium containing the indicated concentration of BAY598 and 1.5% Avicel RL-591 (IFF Nutrition & Biosciences, Oestgeest, The Netherlands) was applied to the cells. Two days post-infection, the overlay was removed, cells were washed with PBS and fixed with 4% PFA in PBS. To visualize YFP-expressing plaques, an Advanced Fluorescence Imager (Intas Science Imaging, Göttingen, Germany) was used.

Lysates from transfected 293T-DSP-mix cells were prepared in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer, boiled at 95 °C for 10 min and sonicated for 1 min. Proteins were separated through SDS-PAGE using 12.5% polyacrylamide gels followed by transfer onto nitrocellulose blotting membranes (Cytivia, Amersham Biosciences Europe GmbH, Freiburg, Germany). Chemiluminescence detection was performed according to the manufacturer’s protocol (ECL Western blotting detection kit; Amersham Biosciences Europe GmbH) by using an INTAS advanced fluorescence imager (INTAS Science Imaging Instruments GmbH, Göttingen, Germany).

Cells were lysed in NP-40 lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% NP-40, Halt Protease Inhibitor). Lysates were quantified by Bradford assay (Carl Roth). In general, 30 μg per sample were separated by SDS-PAGE, transferred onto PVDF membranes and probed with different primary antibodies. Endogenous SAMHD1 was probed with anti-SAMHD1 (3F5) antibody (novusbio); phosphorylated T592 was detected with a pT592-SAMHD1-specific antibody (ProSci). Myc-tagged proteins were probed with an anti-myc (9B11) antibody (Cell Signaling), FLAG-tagged proteins with anti-FLAG M2 antibody (Sigma), T7-tagged proteins with anti-T7 antibodies (Novagen, Abcam), and HA-tagged proteins with an anti-HA (16B12) antibody (Biolegend). To control for equal loading of cell lysates membranes were probed with anti-HSP90 α/β antibody (Santa Cruz). Subsequently, PVDF membranes were incubated with anti-mouse or anti-rabbit HRP-labeled secondary antibodies (Cell Signaling) and visualized using HRP substrate on an Intas Advanced Fluorescence Imager (Intas).

In order to verify the expression of each Fc-fusion protein, 20µL of cell supernatants were mixed with 20 µL of 2x SDS loading dye in presence or absence of β-mercaptoethanol (β-Me) and boiled for 2min at 95°C. Samples were loaded onto 8% or 10% SDS-PAGE gels and run at 120V for 1h15. The separated proteins were then transferred onto nitrocellulose membranes (GE Healthcare, Healthcare, Little Chalfont, UK), followed by an incubation of 1h under rotation with 5% milk in PBS-Tween20 0.1% (=PBS-T) to saturate the membranes. Ag-Fc fusion proteins were then detected in parallel through the Fc-portion, as well as their corresponding antigen to confirm, the formation of the fusion proteins. For that purpose, the Fc-portions were detected with an anti-mouse IgG coupled to Horseradish Peroxidase (HRP) (Dianova, Hamburg, Germany) while the membranes were stained in parallel with huIgG1 2G12 (Polymun Scientific, Klosterneuburg, Austria) or huIgG1 RGN10933 (18 (link)) antibodies followed by an incubation with an anti-human IgG coupled to HRP (Dianova, Hamburg, Germany) to detect gp120 or RBD, respectively. Subsequent to washes with PBS-T, target proteins were finally detected by the addition of Enhanced Chemiluminescence (ECL) reagent and pictures were captured by the Intas advanced fluorescence imager (Intas Science Imaging, Göttingen, Germany).

Cells were lysed in NP40 lysis buffer (10 mM TrisHCl, 150 mM NaCl, 2 mM EDTA, 0.5 % NP-40, Halt Protease Inhibitor). Lysates were quantified by Bradford assay (Carl Roth). In general, 30 μg per sample were separated by SDS-PAGE, transferred to PDVF membranes and probed with mouse anti-SAMHD1 MAb 3F5 (Origene), rabbit anti-pSAMHD1 T592 (ProSci), or mouse anti-HSP90 MAb (Santa Cruz). The membranes were then probed with anti-mouse or anti-rabbit horseradish peroxidase (HRP)-labeled secondary antibodies (GE-Healthcare) and visualized using HRP substrate (Pierce) on an Intas Advanced Fluorescence Imager (Intas). For CDK inhibitor studies, cycling THP-1 KO cells expressing isoform 1 of muSAMHD1 were incubated with DMSO or one of the CDK inhibitors roscovitine, olomoucine II, or CDK2-specific inhibitor (Calbiochem) at 0.3 µM or 3.0 µM for 14 h prior to cell lysis. For Phos-tag analysis, cells were lysed in EDTA-free NP40 lysis buffer. Samples were separated on 8 % polyacrylamide gels containing 25 µM Phos-tag Reagent (Wako Pure Chemicals) and 100 µM MnCl₂. Upon washing the gels with transfer buffer containing additional 10 mM EDTA, proteins were transferred onto PDVF membranes. Membranes were probed with mouse anti-myc (Santa-Cruz) and anti-mouse horseradish peroxidase (HRP)-labeled secondary antibodies (GE-Healthcare).