Alexa546 conjugated antibodies

CD4⁺ T cells resuspended in 1% protein inhibitor mix (Sigma-Aldrich) were lysed by repeated freezing in liquid nitrogen and thawing. After adding 20× PBS lysates were spun at 2000xg for 3 min. The supernatant was centrifuged at 18,000xg for 20 min. The pellet was taken up in 100 mM NaCl, 50 mM HEPES (pH 7.4), 1% protein inhibitor mix, and 0.5% Triton-X-100 (Carl Roth, Karlsruhe, Germany). Proteins were separated on 10% NuPAGE Bis-Tris gels (Invitrogen) and blotted onto nitrocellulose membranes (Mini-Transblot, Bio-Rad, Munich, Germany) for immunodetection. Primary antibodies were anti-P2RX5 (clone 1C5 for immunoblotting or polyclonal for immunocytochemistry, Abnova, Taipei, Taiwan), anti-talin (clone 8d4, Sigma-Aldrich), anti-GAPDH (clone GAPDH-71.1, Sigma-Aldrich) monoclonal mouse antibodies, and anti-LFA-1 (against integrin, alpha-L, clone EP1285Y, Abcam, Cambridge, UK) monoclonal rabbit antibody. Secondary antibodies were anti-mouse HRP-coupled polyclonal sheep IgGs (Jackson ImmunoResearch, Newmarket, UK) and anti-rabbit HRP-coupled polyclonal IgGs (Jackson ImmunoResearch). CD4⁺ T cells were fixed and immunostained essentially as described [44] (link). Secondary Alexa488- and Alexa546-conjugated antibodies were from Invitrogen. Fluorescence images were taken with a Fluoview Fv1000 confocal microscope (Olympus, Tokio, Japan).

Vero E6 cells were grown to 90% confluency on glass coverslips and infected with rSARS-CoV-ΔE, rSARS-CoV wt, rSARS-CoV-E-N15A and rSARS-CoV-E-V25F at an MOI of 0.3. At the indicated hpi, media were removed and cells were washed twice with PBS and fixed with 4% paraformaldehyde in PBS for 30 min at room temperature. Then, cells were washed twice with PBS and permeabilized for 10 min with 0.2% saponin and 10% FBS in PBS. Primary antibody incubations were performed in PBS containing 10% FBS and 0.2% saponin for 1 h 30 min at room temperature. Immunofluorescence was performed using a mouse mAb specific for ERGIC53 (dilution 1∶200, Alexis Biochemicals), and a rabbit pAb specific for E protein [15] (link) at 1∶2000 dilution. Coverslips were washed four times with PBS between primary and secondary antibody incubations. Alexa 488- or Alexa 546-conjugated antibodies specific for the different species (dilution 1∶500, Invitrogen) were incubated for 45 min at room temperature in PBS containing 10% FBS. Nuclei were stained using DAPI (dilution 1∶200, Sigma). Coverslips were mounted in ProLong Gold anti-fade reagent (Invitrogen) and examined on a Leica SP5 confocal microscope (Leica Microsystems). Colocalization studies were performed using Leica LAS AF v2.6.0 software.

The following commercial antibodies, and the indicated concentrations, were used in this study. C-Myc (#E0115; 1:1000), Chk1 (G-4) (#H2714; 1:1000) and GST (Z-5) (#K0713; 1:1000) were purchased from Santa Cruz Biotechnology. M2 anti Flag Mouse antibody (#SLBT7654; 1:5000), cdc27 (AF3.1) (1:1000) and Actin (#087M4850; 1:10,000) were purchased from Sigma. Cdh1 (#CC43-100UG; 1:500) was purchased from Calbiochem. Cyclin A2 (BF683) (#6; 1:1000), βTRCP1 (D13F10) (1:1000) and Phospho-Chk1Ser345 (133D3) (#15; 1:1000) were obtained from Cell Signaling. HA (#SJ254200; 1:1000) antibody was purchased from Biolegend. Plk1 (3F8) (#06050819; 1:500) was obtained from Enzo Life Sciences. HA antibody (HA.C5 #18181) (1:1000) was purchased from Abcam. Secondary antibodies for western blotting were purchased from LI-COR Biosciences. Anti-phospho-Histone H2AX (clone JBW301) (#2977883, 1:500) was purchased from EMD Millipore Corp. Alexa546-conjugated antibodies (#A11030) for immunofluorescence were purchased from Invitrogen.