Anti rabbit igg alexa flour 488

TNFα (210-TA) (for in vivo experiments) was from R&D system; D-GalN (101743) was from Millipore; ConA, CHX, puromycin and doxycycline (DOX) were from Sigma Aldrich; ZVAD-FMK (s7023) and sp600125 (s1406) were from Selleck; LCL161 (HY-15518) was from MCE; TNFα (300-01 A) (for in vitro experiments) was from Peprotech; Flag-TNF was from Enzo Life (ALX-522-009-C050); Lipofectamine 2000 Transfection Reagent was from Invitrogen. The following antibodies were used for flow cytometry: FITC-Annexin V (556420), 7-AAD (559925) were from BD Biosciences; Alexa Flour^® 488 anti-rabbit IgG was from Invitrogen. The following antibodies were used for immunoprecipitation and immunoblotting: anti-Bcl-3 (sc-185), anti-Caspase 8 (sc-6136), anti-STAT3 (sc-482), anti-UB (sc-8017), anti-rabbit IgG (sc-2027) and anti-mouse IgG (sc-2025) were from Santa Cruz; anti-Caspase 8 (9746), anti-cleaved Caspase 3 (9661), anti-pJNK (4668), anti-JNK (9252), anti-pIκB (2859), anti-PARP (9542), anti-FADD (2782), anti-A20 (5630), and anti-CYLD (8462) were from Cell Signaling Technology; anti-HA tag (51064) and anti-MYC tag (60003) were from Proteintech; anti-cIAP1/2 (MAB3400) was from R&D; anti-pSTAT3 (CY6566) was from Abways; anti-RIP1 (610458) was from BD Biosciences; anti-β-Actin (A2228), anti-Flag (F3165) and anti-Flag M2 affinity gel (A2220) were from Sigma Aldrich; anti-GAPDH (KC-5G4).

For WB detection, we used the following primary antibodies: mouse anti-V5 monoclonal antibody (46-0705, Invitrogen), mouse anti-FLAG M2 monoclonal antibody (F3165, Sigma-Aldrich), rabbit anti-HA polyclonal antibody (H6908, Sigma), rabbit anti-SENP1 polyclonal antibody (ab108981, Abcam), mouse anti-GAPDH monoclonal antibody (AM4300, Invitrogen), goat anti-GAPDH antibody (NB-300-320, Novus biologicals), mouse anti-MYB (5E11) antibody (ab10934, Abcam), and rabbit anti-MYB H141 (sc7874, Santa Cruz Biotechnology). The following secondary antibodies were used for WB: anti-rabbit IgG-HRP (711-03-152, Jackson Immuno-Research), anti-mouse, anti-goat and anti-rabbit IRDye 680 RD (926-68072, 926-68074, and 926-68073, respectively), and anti-mouse IRDye 800 CW (926-32212) (LI-COR).
For immunoprecipitation, we used the following antibodies: FLAG M2 magnetic beads (Sigma), protein G Dynabeads (10004D, Invitrogen), rabbit anti-HA (H6908, Sigma), and mouse anti-V5 monoclonal antibody (46-0705, Invitrogen).
For immunofluorescence, we used the following antibodies: mouse anti-V5 monoclonal antibody (46-0705, Invitrogen), rabbit anti-SENP1 polyclonal antibody (ab108981, Abcam), Alexa Flour 488 anti-rabbit IgG (Invitrogen), and Alexa Flour 647 anti-mouse IgG (Invitrogen).

Neutralizing antibodies were evaluated in all the individuals tested as described previously^{7 (link)}. In brief, inactivated serum samples (56 °C, 30 min) were first diluted 1:10 followed by 1:2 serial dilutions. Then, 50 µl of the diluted sera was mixed with 50 µl of a SARS-CoV-2 Wuhan-Hu-1 dilution with a concentration of 4,000 TCID₅₀ ml⁻¹ and incubated for 1 h at 37 °C. Serum-virus mix was added to a monolayer of Vero cells and further incubated at 37 °C, 5% CO₂. After 8 h of incubation, cells were fixed using 4% paraformaldehyde (PFA) and incubated for 30 min at room temperature. Then, PFA was removed and cells were incubated for 15 min with 80% methanol. Furthermore, plates were blocked using 1% BSA in PBS-0.05% Tween 20 for 30 min at 37 °C. SARS-CoV-2 was detected using a 1:1,000 dilution of rabbit polyclonal anti-SARS-CoV-2 nucleocapsid (SinoBiological). After 1 h incubation at 37 °C, cells were washed with PBS-0.05% Tween 20 and incubated with a 1:1,000 dilution of anti-rabbit-IgG-Alexa Flour 488 (Invitrogen). Finally, cells were washed twice with PBS-0.05% Tween 20. Fluorescent cells were counted using the CTL S6 Ultimate-V Analyzer and data were analyzed using the CTL ImmunoSpot version 7.0.20.0 software. Neutralizing antibody titers are expressed as the dilution that gave a 50% reduction of stained cells.