Azure c300 digital imaging system

Cell lysates were collected by adding RIPA buffer with protease and phosphatase inhibitors (Thermo Scientific, PIA32959, PIA32957) to BMDM treated or infected as indicated. 4x SDS loading dye was then added to samples, which were boiled for 20 min and resolved by SDS-PAGE and then transferred to PVDF membranes. Western blotting for caspase-1 (caspase-1 p45 and p20), phosphorylated IκBα and total IκBα were then performed by incubating membranes in primary antibody diluted in 5% milk in TBST overnight at 4 °C (See Table 1for a list of antibodies). The next day, membranes were washed 3x in TBST buffer and incubated for 45 min in secondary antibody diluted in 5% milk in TBST (Table 1). Membranes were washed again, and images obtained using Super Signal West Fempto substrate (ThermoFisher, A53225) and an Azure C300 digital imaging system.Table 1

Antibodies.

Table 1

Antibodies	Catalog Number
Rabbit anti-mouse β-Actin	Cell Signaling Technology, 8457S
Rabbit anti-mouse IкB-α	Cell Signaling Technology, 9242S
Rabbit anti-mouse Phospho-IκB-α	Cell Signaling Technology, 2859S
Mouse anti-mouse Caspase-1	Adipogen, 661,228
Anti-Rabbit-HRP secondary	Jackson Immuno Res.111−035-144
Anti-mouse-HRP secondary	BioRad, HAF007

For SDS-PAGE, total protein lysates were prepared using saponin-lysed parasites resuspended with 1X Laemmli loading buffer diluted in 1x PBS supplemented with 1X Roche Complete protease inhibitors cocktail. Protein samples were separated in 4–15% polyacrylamide gels using Precision Plus Protein Standards as MW ladder (Bio-Rad) and transferred to 0.2 μm Immobilion-P^SQ transfer membrane (Millipore, Cat. No ISEQ00010) using a Bio-Rad transfer system. Membranes were blocked in 5% skim milk/1x TBS-Tween20 for 1 hour at RT. Primary and secondary antibodies were prepared in 3% skim milk/1x TBS-Tween20 and incubated for 1 hour at RT. Membranes were washed four times with 1x TBS-Tween20 for 10 min, after primary and secondary antibody incubations. The following primary antibodies were used in this study: Anti-Ty1 BB2 mouse (1:2,500; Invitrogen Cat. N^o MA5–23513), anti-PhIL1 rabbit (1:5,000 (46 (link))), anti-PfHsp70 rabbit (1:5,000; StreesMarq Biosciences Cat. N^o SPC-186D), anti-Histone 4 rabbit (1:2,000; Diagenode Cat. N^o C15410156–50). HRP-conjugated anti-mouse and anti-rabbit antibodies were used (1:5,000, Millipore). Immunoblots were incubated with the chemiluminescent substrate SuperSignal West Pico PLUS (ThermoFisher, Cat. N^o 34578) following manufacturer directions. Chemiluminescent images were obtained using an Azure c300 digital imaging system (Azure Biosystems).