Ab175772

Protein extraction was done with RIPA lysis buffer (150 mM NaCl, 1% IGEPAL CA-630, 0,5% sodium deoxycholate, 0,1% SDS, 50 mM Tris, pH 8.0) containing 1 × protease/phosphatase inhibitors. Samples were lysed in the TissueLyser (Qiagen) until the tissue was fully homogenised. Samples were incubated on ice for 30 min, followed by a centrifugation step (30 min, 4 °C, 18,000 g). The supernatant was collected, and centrifuged again (20 min, 4 °C, 18,000 g). Then, supernatant was used to quantify the protein concentration with a Bradford assay (B6916, Sigma). The following primary antibodies were used: anti-tyrosine hydroxylase (ab137869, Abcam, diluted 1:2000), anti-glycerol kinase (ab126599, Abcam, diluted 1:2000) and anti-alpha tubulin (sc-23948, Santa Cruz, diluted 1:2000). The following secondary antibodies were used: goat anti-rabbit IgG, HRP (ab6721, Abcam, diluted 1:10,000), goat anti-mouse IgG, HRP (AP130P, Sigma, diluted 1:10,000), mouse IgG AF790 (ab175782, Abcam, diluted 1:10,000) and rabbit IgG AF680 (ab175772, Abcam, diluted 1:10,000). Immunoblots were visualised with chemiluminescence for HRP-linked secondary antibodies (Clarity™ Western ECL Substrate, 1,705,060, BioRad) or with fluorescence for the Alexa Fluor labelled secondary antibodies.

Cells on slides were fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature and were permeabilized with 0.1% Triton X-100 in 1 × PBS for 5 min and blocked with blocking buffer (1% BSA and 2% donkey serum diluted in PBS) for 30 min. Immunofluorescence analyses of SARS-CoV-2-infected cells were performed using a rabbit anti-SARS-CoV-2 spike S/S2 protein antibody (1:500, 40590-T62, S&B), a mouse anti-dsRNA antibody (1:200, 10010200, J2-1909, SCICONS), an anti-ACE2 antibody (1:100, A12737">A12737, Abclonal), IRF-3 (D6I4C) XP rabbit mAb (1:300, #11904, CST), Alexa Fluor 680 donkey anti-rabbit IgG (H+L) (1:1000, ab175772, Abcam), and Alexa Fluor 555 donkey anti-mouse IgG (H+L) (1:1000, ab150106, Abcam). All cells were mounted with ProLongTM Gold Antifade with DAPI (Life Technologies, P36931) and imaged with a TissueFAXS 200 flow-type tissue cytometer (TissueGnostics GmbH, Vienna, Austria). All statistical analyses of immunofluorescence staining present the results from at least 3000 cells per replicate, and data are shown as the mean ± s.e.m.