Odyssey fc imaging system

Dot blot analysis was used to detect specific interactions of the various MAbs with the sub-fragments of BmGPI12 or individual peptides (11 amino acids each). Samples (100 ng) were spotted on a nitrocellulose membrane and dried at room temperature for 1h. The membrane was then blocked by application of 200 µl of blocking buffer (5% BSA in TBS with 0.05% Tween-20 (TBST-0.05%)) (Singh et al., 2022 (link)) into each well for 1h at room temperature. Following this step, the blocking buffer solution was replaced with 200 µl of 0.1% BSA in TBST-0.05% buffer and kept for 1h at room temperature or overnight at 4°C. Primary antibodies were diluted 1:1000 in 0.1% BSA in TBST-0.05% buffer and added to the membranes for 1h at room temperature, followed by incubation with HRP-conjugated goat anti-mouse IgG (1: 5,000 dilution) (31466, Thermo Fisher) secondary antibody for 45 min at room temperature. The blots were washed three times with TBST-0.05%, once with PBS (5 min), and then developed using the Super signal™ West Pico PLUS chemiluminescent substrate (34577, Thermo Scientific) and imaged using the Odyssey Fc imaging system.

Neutrophils (5 × 10⁶) were treated with 10 µM TX and its metabolites (N‐desmethyltamoxifen and endoxifen) or vehicle (DMSO 0.2%) and then incubated at 37°C for 0, 5, 10, 20, 30 and 60 min. The cells were lysed, and 60 µg of purified protein was analysed by 12% SDS‐page and transferred to a nitrocellulose membrane. The primary antibody against procaspase and caspase‐3 (Cell Signaling) was incubated for 1 hr. The membranes were developed using Super Signal West Femto (Thermo Scientific) and visualized on the Odyssey Fc Imaging System. Each membrane was re‐probed with anti‐vinculin antibody (Santa Cruz).