S6508

For pharmacological inhibition, cells were treated using the following concentration of drugs prior to sample fixation or laser ablation experiment: sodium orthovanadate (S6508, Sigma) 100 μM, RK682 (RK2033, Sigma) 10 μg/ml; ALLN (ab141445, Abcam) 10 and 50 μM, ALLM (ab141446, Abcam) 10 and 50 μM, nocodazole (M1404, Sigma) 10 μM, and Y-27632 (Y0503, Sigma) 10 μM.
While sodium orthovanadate is a generic alkaline and tyrosine phosphatase inhibitor, RK682 is a specific and non-competitive inhibitor of protein tyrosine phosphatase with confirmed low micromolar inhibitory activity against protein tyrosine phosphatases CD45 and PTP1B, and dual-specificity phosphatases VHR, CDC-25A-B-C (Hamaguchi et al., 1995 (link)); nevertheless, CD45 and VHR genes are not expressed in MDCK and EpH4 cells, as indicated by RNAseq data (Supplementary Table 1), while inhibition of CD25 requires longer incubation period (20 h) with respect to what we have used in the current paper (1 h).
ALLN and ALLM are inhibitors for calpains, with high binding efficiency for calpain-1 and calpain-2, respectively. ALLN has been reported to inhibit the calpain/PTP1B axis in endothelial cells (Zhang et al., 2017 (link)).

A total of 5 × 106 PBMCs were washed twice with 1X PBS and centrifuged at 1500 rpm for 5 min. Cells were lysed in a 1X RIPA buffer containing 150 mM NaCl, 1% NP-40 (Sigma Aldrich; NP40S), 50 mM Tris-HCl, pH 8, 0.5% deoxycholic acid (Sigma Aldrich, D6750), 0.1% SDS (Sigma Aldrich, 71736), protease (Sigma Aldrich; S8830) and phosphatase inhibitors (Sodium Orthovanadate; Sigma Aldrich; S6508) (Sodium Fluoride; Sigma Aldrich; S7920). Then, 15 µg of protein lysates was subjected to electrophoresis on 4–20% SDS-PAGE gradient gels (NuSep, Germantown, MD, USA; NN12-420), according to the manufacturer’s instructions. Then, the gels were transferred to nitrocellulose membranes (Bio-Rad, 162-0115) for 2 h in Tris-glycine buffer. The membranes were blocked in PBS-0.1% Tween 20 solution containing 3% BSA, probed with specific antibodies, and developed using ECL Blotting Substrate (Advansta, San Josè, CA, USA; K-12045-D20).

Blood was processed into serum that was heat-inactivated at 56°C. Nonidet-P40 (NP40) non-ionic detergent was added to saliva samples to a final concentration of 0.05% (vol/vol), both to inactivate SARS-CoV-2 and as a preservative [15 (link)]. Saliva extracts were sterilely passed through a 22 μm filter, before addition of protease inhibitors to inhibit sample degradation. Inhibitor sources and final concentrations were Aprotinin, 8.5 μg/mL (Sigma Aldrich); phenylmethanesulfonyl fluoride, 5.9mM (Sigma-Aldrich 93482); and sodium orthovanadate, 1.2mM (Sigma-Aldrich S6508). Processed saliva samples were stored at −20°C [16 (link)]. In pilot experiments, we confirmed that NP40 and the aforementioned protease inhibitors had no effect on anti-S-protein titers derived using heat-inactivated saliva samples or the CR3022 anti-RBD MAb. The antibody titers were also unaffected when saliva samples were subjected to 3 freeze/thaw cycles.

Cells were harvested on ice, washed twice with cold PBS, collected, lysed in 50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1 mmol/L EDTA, 1% TritonX-100 (X100; Sigma) supplemented with protease and phosphatase inhibitors (sodium orthovanadate [S6508; Sigma], 1 mmol/L DTT, 2 mmol/L NaF [S1504; Sigma and complete Mini Protease Inhibitor Cocktail [ROCHE]). Western blotting was conducted using standard methods and as previously described 36 (link) with the following primary antibodies: NR2F2 (ab41859, 1/1000, Abcam), NR2F6 (ab137496, 1/1000, Abcam), SMAD1 (#9743, 1/1000, Cell signaling). Protein ladder was the Lonza ProSieve™ QuadColor™ Protein Mark.

Cells were seeded (1 × 10⁶ cells/flask) and allowed to attach for 24 h. The medium was then replaced by serum free medium and following 24 h the samples were collected on ice as following: First the medium was transferred and proteases and phosphatases inhibitors (protease inhibitors cocktail (1:100, 539134, Calbiochem, San Diego, CA, USA), PMSF (2 mM, P7626, Sigma) and sodium orthovandate (1 mM, S6508, Sigma)) were added to the medium. The cells were washed with cold PBS, RIPA lysis buffer (in mM: 50 Tris-HCl pH 8.0, 150 NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, and 0.1% SDS) containing proteases and phosphatases inhibitors was added and the cells were scraped and collected. The collected medium was transferred to centrifugal filter device 10 K (UFC901008, Merck–Millipore, Molsheim, France), centrifuged (4000 g, 20 min), and the concentrated sample was collected for further analysis. The cells incubated on ice for 10 min, centrifuged (16000 g, 20 min), and the supernatant was collected for further analysis.