Sc 73614

Protein lysates were boiled for 5 minutes at 95°C in SDS protein buffer (5x laemmli sample buffer, Thermo Fisher Scientific) and separated by SDS-PAGE following transfer to a polyvinylidene fluoride (PVDF) membrane. Primary antibodies were anti-alpha-Amylase (rabbit polyclonal, CST-4017, Cell signaling, Boston, USA), anti-Pancreatic-Lipase-A3 (mouse monoclonal, SC-374612, Santa-Cruz, Texas, USA), anti-Glyoxalase-I (Glo-I, mouse monoclonal, SC-133214, Santa-Cruz), anti-NF-ĸB (p65 subunit, mouse monoclonal SC-8008, Santa-Cruz) and anti-Vinculin (mouse monoclonal, SC-73614, Santa-Cruz). Secondary antibodies were anti-mouse (goat anti-mouse, 1858413, Pierce / Thermo Fisher Scientific) and anti-rabbit (goat anti-rabbit, 1858415, Pierce). Western Blot signals were quantified using imager (G-Box Chemie XX9, Syngene, Cambridge, UK). Signals were normalized to their respective loading controls using ImageJ-Software (v. 1.48, http://imagej.nih.gov) and: GeneTools (Syngene).

Protein extraction and immunoblotting were carried out as indicated (Pirone et al., 2019 (link); Ungaro et al., 2012 (link)). Antibodies against ZMAT3 (ab191536, Abcam), P53 (sc‐126, Santa Cruz), and Vinculin (sc‐73614, Santa Cruz) were used for protein detection.

Primary antibodies from Cell Signaling Technology (Danvers, MA, USA) were used to detect Caspase 3 (#9662; 1:1000/1:500 [full-length/cleaved]), Bad (#9239; 1:500), phospho-Bad (Ser112) (#5284; 1:500), Erk (#4695; 1:1000), phospho-Erk (#9101; 1:1000), Akt (#9272; 1:1000), phospho-Akt (#4060; 1:200), MK2 (#3042; 1:500), phospho-MK2 (Thr222) (#3316; 1:200), phospho-MK2 (Thr334) (#3007; 1:200), p38 (#9212; 1:1000), phospho-p38 (#4511; 1:2000), JNK (#9252; 1:1000), phospho-JNK (#9251; 1:500), c-Jun (#9165; 1:1000) and phospho-c-Jun (#3270; 1:1000). Primary antibodies against p53 (sc-47698; 1:1000), phospho-p53 (Ser392) (sc-51690; 1:500) and Vinculin (sc-73614; 1:1000) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Polyclonal antibodies against TCRV (1:1000) and JUNV (1:1000) NP (produced in guinea pigs [15 (link)]) were used to confirm viral infection. Fluorescently labeled secondary antibodies were purchased from LI-COR (IRDye 680RD Donkey anti-Mouse IgG, IRDye 680RD Donkey anti-Guinea Pig IgG, IRDye 800CW Donkey anti-Rabbit IgG) and used at a dilution of 1:15000. A goat anti-rabbit IgG HRP-linked secondary antibody (#7074; 1:5000, Cell Signaling Technology) was used for the detection of Bad.

We used the following antibodies: A300-516A (Thermo Fisher), polyclonal anti-DNA-PKcs 1:5000; EPR2302 (GeneTex, Inc), monoclonal anti-FANCD2 1:2000; T5168 (Merck), monoclonal anti-αTubulin 1:8000; sc73614 (Santa Cruz Biotechnology), monoclonal anti-Vinculin 1:1000; A0545 (Merk), HRP (horseradish peroxidase)-conjugated anti-rabbit IgG 1:10 000; A0168 (Merk), HRP-conjugated anti-mouse IgG 1:10 000.

NP and NP mutants were expressed in HEK 293T cells in 12-well format by transfecting 500 ng of the respective expression plasmid with TransIT LT-1 (Mirus Bio LLC) as recommended by the manufacturer, with 3 µl transfection reagent per µg DNA. One day after transfection, medium was exchanged to DMEM5 and samples were harvested on the following day. For this, cells were resuspended in 1 ml PBS, spun down for 5 min at 800 x g, and then boiled for 5 minutes at 95°C in 1x SDS-sample buffer. SDS-PAGE and semi-dry Western blotting was done as described before [26 (link)]. For analysis of expression either a polyclonal antibody against NP (rabbit, Gentaur, 0301-012) (for the IDR mutants) or a polyclonal antibody against c-myc (rabbit, Thermo Fisher, PA1-981) (for the point mutants) was used at a dilution of 1:5.000. As a loading control, a monoclonal antibody against Vinculin was used (mouse, Santa Cruz, sc-73614, 1:1.000). Secondary antibodies were goat anti-rabbit IgG IRDye 800CW (Li-cor, 926-32211) and goat anti-mouse IgG IRDye 680RD (Li-cor, 926-68070) at a dilution of 1:15.000. Blots were imaged with the Odyssey CLx system (Li-cor).

Protein lysates were broiled for 5 min at 95 °C in SDS protein buffer (5 × laemmli sample buffer, Thermo Fisher Scientific) and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) following transfer to a polyvinylidene fluoride (PVDF) membrane (both Roth, Karlsruhe, Germany). Primary antibodies against Glo-I (SC-133214), NF-ĸB (p65 subunit, mouse, anti-mouse, -rat, -human, monoclonal SC-8008, Santa-Cruz), RAGE (SC-365154), β-Actin (mouse, anti-mouse, -rat, -human and other, C-15, A5441, Sigma-Aldrich), PPIB (anti-cyclophilin-B, rabbit, anti-mouse, polyclonal, ab16054, abcam, Cambridge, United Kingdom) and Vinculin (mouse, anti-human, -mouse, -rat, -avian, monoclonal, SC-73614, Santa-Cruz) were used. Vinculin was used as a housekeeping marker for Western blot as it was not influenced by CN (Supplementary Figure S4). Secondary antibodies were anti-mouse (goat, anti-mouse, polyclonal 1858413) and anti-rabbit (goat, anti-rabbit, 1858415, polyclonal, both Pierce/Thermo Fisher Scientific). Western blot lanes were quantified using an imager (G-Box Chemie XX9, Syngene, Cambridge, UK). Signals were normalized to their respective loading controls using ImageJ Software (v. 1.48, http://imagej.nih.gov, accessed on 13 August 2021) and GeneTools (Syngene, Frederick, ML, USA)