Chemiluminescence western blotting kit

Unless otherwise noted, cells of the late exponential phase were harvested, washed with phosphate buffered saline (PBS), resuspended in the same buffer, and sonicated. Protein concentrations of the cell lysates was determined by the bicinchoninic acid assay (Pierce Chemical). For heme-staining, the cell lysates were separated on SDS-PAGE using 12% polyacrylamide gels and stained with 3,3′,5,5′-tetramethylbenzidine (TMBZ) as described elsewhere43 (link). Immunoblotting analysis was performed essentially the same as previously described44 (link). Proteins separated on SDS-PAGE were electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane according to the manufacturer’s instructions (Bio-Rad). The gels were blotted for 2 h at 60 V using a Criterion blotter (Bio-Rad). The blotting membrane was probed with rabbit polyclonal antibodies against NrfA. The goat anti-rabbit IgG-HRP (horseradish peroxidase) (Roche Diagnostics) was used as the secondary antibody (1:5,000) and the signal was detected using a chemiluminescence Western blotting kit (Roche Diagnostics) in accordance with the manufacturer’s instructions. Images were visualized with a UVP imaging system.

Western blotting analysis was performed for detection of His₆-tagged proteins as previously described (Dong et al., 2012 (link)). Cells entering the stationary phase were harvested by centrifugation, washed with PBS (pH 7.0), resuspended in the same buffer, and sonicated. The cell lysates containing the same amount of proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE, 10%). Proteins were then electrophoretically transferred to polyvinylidene difluoride membranes according to the manufacturer’s instructions (Bio-Rad). The gels were blotted for 2 h at 60 V using a Criterion blotter (Bio-Rad). The blotting membrane was probed with a rabbit polyclonal antibody against His₆-tag. Goat anti-rabbit IgG-HRP (horseradish peroxidase) (Roche Diagnostics) was used as the secondary antibody (1:5,000) and the signal was detected using a chemiluminescence Western blotting kit (Roche Diagnostics). Images were visualized with a UVP imaging system.

CAR T‐cell lysates were prepared using lysis buffer containing 150 mM NaCl, 50 mM Tris‐HCI (pH 7.4), 2% Nonidet P‐40, 2 mM CaCl2, and protease and phosphatase inhibitor cocktails (Roche). When indicated, 5 mM EDTA was added to the lysis buffer as negative control condition. A sum of 5 × 10⁶ cells were pelleted per condition, washed with PBS, and lysed in 500 μL of lysis buffer. Cell lysates were incubated on ice for 15 min and centrifuged at 12 000 g for 10 min. Cell lysates were then precleared overnight using protein G‐agarose beads (Roche) and incubated for 2 h at 4°C with 3 μg of murine E‐selectin‐human Fc chimera (“E‐Ig,” R&D Systems), as described.¹⁷ Agarose beads were then washed twice with lysis buffer, and immunoprecipitated glycoproteins were collected by boiling the beads in the presence of 2‐mercaptoethanol in Laemmli loading buffer. For western blot (WB) analysis, immunoprecipitates were resolved on a 7.5% SDS‐PAGE gel (Bio‐Rad Laboratories) and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio‐Rad). The membrane was then blocked with blocking reagent (Chemiluminescence Western Blotting Kit, Roche), and incubated with monoclonal antibodies (MoAb) against PSGL1 (clone KPL1, BD), CD43 (clone IG10, BD), and CD44 (clone 2C5, R&D). Protein bands were detected by chemiluminescence using Lumi‐Light substrate (Chemiluminescence Western Blotting Kit).