Roti block

Immunoblot analyses were performed as described in [9 (link)] with the following modifications. Proteins were transferred onto PVDF membranes over night at 37 V except for keratin 17 immunoblots which were done at 100 V for 100 min. Membranes with bound proteins were blocked for 1 h in 10% (v/v) Roti®-Block (Carl Roth) and incubated with primary antibodies in buffer [50 mM Tris, 130 mM NaCl, 0.1% (v/v) Tween 20, pH 7.6] containing 1% or 2% or 10% (v/v) Roti®-Block. The same buffer with 1% (v/v) Roti®-Block was used for secondary antibody incubation. Bound antibodies were detected with AceGlow™ Chemiluminescence Substrate (Peqlab) on a Fusion-Solo.WL.4M with Fusion-Capt Advance Software 16.06 (Vilber Lourmat). Protein mass was determined with the ProSieve QuadColor Protein Marker (Lonza). Unprocessed immunoblots and protein transfer controls are included as S1 Files.

Cell lysates were prepared with NP-40 lysis buffer (10% [v/v] glycerol; 2 mM EDTA, pH 8; 137 mM NaCl; 50 mM Tris pH 7.2; 0.5% [v/v] NP-40) supplemented with 20 mM sodium fluoride, 2 mM phenylmethanesulfonyl fluoride, and 2 mM sodium orthovanadate. After incubation for 20 min on ice, lysates were cleared by centrifugation at 15,900 × g for 20 min at 4°C. Subsequently, 4× Laemmli buffer (125 mM Tris-HCl pH 6.8, 4% SDS, 20% [v/v] glycerin, bromophenol blue, 10% [v/v] 2-mercaptoethanol) was added, and samples were boiled at 95°C for 5 min. After separating the proteins by SDS-PAGE, Western blotting on a nitrocellulose membrane (GE Healthcare) was performed. Membranes were blocked with 1× RotiBlock (CarlRoth) and incubated with the indicated primary antibodies in 1× RotiBlock at 4°C overnight. The following primary antibodies were used: CYTIP and cytohesin-1 (mAbs rat anti-CYTIP clone 2F9 and rat anti-cytohesin-1 clone 7H2, respectively), anti-IE2 (clone 12E2, Santa Cruz Biotechnology), anti-ICP0 (clone 11060, Santa Cruz Biotechnology), and anti-GAPDH (clone 6C5, Millipore). After incubation with the appropriate secondary HRP-labeled antibody (Cell Signaling Technology) for 1 h at room temperature, signals were detected using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare).

Cell lysis was performed in BEX buffer (25mM Tris at pH 8.0, 20mM NaCl, 0.6% w/v deoxycholate and 0.6% Igepal CA-630). Gel electrophoresis was run under denaturing conditions with NuPage Novex Bis-Tris 4%-12% gradient gels (Invitrogen) and MOPS running buffer (Invitrogen). Semi-dry protein transfer on polyvinylidene difluoride membranes was followed by blocking in 1x RotiBlock (Roth) for 1 h. Incubation with primary antibodies was performed overnight at 4°C in 1x RotiBlock. Incubation with horseradish peroxidase-coupled secondary antibodies (1h, room temperature) and subsequent analysis with LAS-4000 (GE) was performed the following day.

Cell lysates of passage 15 (on CD46^-MDBK cells) and passage 0 (on NTC cells) were prepared 72 h after infection of the cells. For this purpose, cells were harvested and incubated for 30 min on ice in lysis buffer (Na₂HPO₄, 1% Triton), supplemented with protease inhibitor (Roche). Proteins were separated under nonreducing conditions on a 10% SDS-polyacrylamide gel for 75 min at 130 mA and subsequently blotted onto a GE Healthcare Amersham™ Protan™ nitrocellulose membrane (Thermo Fisher Scientific). The membrane was blocked with Roti^®Block (Carl Roth, Karlsruhe, Germany) for 1 h. For the detection of pestivirus E^RNS, the membrane was incubated with monoclonal antibodies HC/TC169/2/3 and BVD/C46/2/1 for 1 h (1:100 in Roti^®Block, kindly provided by Prof. Becher, Institute of Virology, University of Veterinary Medicine Hannover). The POD-antimouse antibody (Dianova, Hamburg, Germany) was used as a secondary antibody, diluted 1:20,000 in phosphate buffered saline with tween (PBST). Proteins were detected by chemiluminescence, using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific).

After protein isolation, the concentration was measured (Pierce BCA Protein Assay Kit; Thermo Scientific, Schwerte, Germany, 23225) and 20 µg of total protein was used for the electrophoresis on 12.5% (w/v) SDS gels. Gels were transferred onto nitrocellulose membranes (BA 85 6E Protean; Whatman plc/G E Healthcare Life Sciences, Darmstadt, Germany), blocked with Roti-Block (Carl Roth GmbH, Karlsruhe, Germany, A151.2) for 30 min, and incubated overnight at 4 °C with the primary antibody (Table 3) diluted in Roti-Block. Membranes were washed 3 times with washing buffer, incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Table 3) and washed 5 times. The signal detection was performed using Luminata Classico Western HRP Substrate (Merck, Darmstadt, Germany, WBLUCO0500) and was visualized with FusionFX7. β-Actin was used as a reference.

Western blots were performed according to Buck et al. (2019) (link). In short, cells were homogenized in a lysis buffer [50-mm Tris–HCl pH 6.8, 2% SDS, and 1× protease inhibitor (cOmplete; Roche)] with a cell homogenizer (Savant Fastprep FP120; Thermo Fisher) and glass beads. Supernatants according to 1 μg of Chl a were separated on 14% LDS–polyacrylamide gels, blotted on nitrocellulose membranes, blocked with 1× Rotiblock (Carl Roth, Karlsruhe, Germany) and incubated with either an anti-Rubisco antibody (AS03037; Agrisera, Vännäs, Sweden) or an anti-Lhcx antibody, each in 1:10,000 dilution [raised by Buck et al. (2019) (link); AS194367; Agrisera] in 1× Rotiblock. The secondary HRP-labeled goat anti-rabbit antibody (AS09602; Agrisera) was either applied in 1:10,000 (Lhcx) or 1:20,000 (Rubisco) dilution in 1× Rotiblock. Detection was performed in an Odyssey FC (LI-COR) with Roti-Lumin Plus (Carl Roth) as substrate.

CD8+ T cells were collected, washed twice in ice cold PBS and lysed in UTB buffer with β- mercaptoethanol. Protein lysates were quantified with Bradford Assay reagent (BioRad) and separated by SDS-PAGE on Nu-Page 3-8% Tris acetate or 4-12% Bis-Tris gels (Life Technologies). Proteins were transferred to nitrocellulose membranes and blocked with Roti- Block (Carl Roth). Membranes were incubated with primary antibodies (FIH, 1:1000, Santa- Cruz, sc-271780; Tubulin, 1:10000, Abcam, ab6160) overnight at 4°C and then with infrared dye-conjugated secondary antibodies at room temperature. Imaging of membranes was carried out using an Odyssey imaging system (LICOR).