Anti kdel

Protein, 20–30 µg, was separated by electrophoresis on 10–12% SDS-polyacrylamide gels at 60–100 mV and then transferred onto polyvinylidene fluoride (PVDF) membranes for 90 min on constant 200 mA at 4 °C. After protein transfer, membranes were blocked in 5% BSA + Tris-buffered saline plus Tween-20 (TBST) blocking buffer for an hour, then incubated with the respective primary antibodies overnight at 4 °C. Following 3 washes with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Promega,#W401B, Madison, WI, USA) or HRP-conjugated anti-mouse IgG secondary antibodies (BioRad, #170-6516, Mississauga, ON, Canada) (1:5000) for 1 h at room temperature. Signals were detected using ECL plus kit reagents (Perkin Elmer, Guelph, ON, Canada) on a Chem Doc imager (BioRad, Mississauga, ON, Canada). Primary antibodies used were: Anti-Caspase-12 (Abcam, #ab62484, Toronto, ON, Canada); Anti-KDEL (Abcam, #ab176333, Toronto, ON, Canada), which recognizes GRP94, GRP78, and PDI; Anti-XBP1(Abcam, #ab37152, Toronto, ON, Canada); Anti-ATF6 (Proteintech, #24169-1-AP, Rosemont, IL, USA); Anti-PERK (Proteintech, #20582-1-AP, Rosemont, IL, USA); Anti-IRE1α (Proteintech, #27582-1-AP, Rosemont, IL, USA).

We used the following antibodies: anti-TOMM20 (Abcam, USA, cat. ab56783); anti-GM-130 (Abcam, USA, cat. ab52649); anti KDEL (Abcam, USA, cat. ab12223). Control and treated macrophages seeded on chamber slides (0.4 × 10⁶/slide) were fixed in 4% formaldehyde in PBS with 0.05% Triton X-100. After washing in PBS–Tween 20, fixed cells were blocked in casein blocking buffer (Bio-Rad, USA) with 0.05% Tween 20 and subsequently incubated in blocking buffer with a 1:200 dilution of primary antibodies and rhodamine–phalloidin overnight at 4 °C. Subsequently, after extensive washing in PBS–Tween 20, cells were mounted in ProLong^® Diamond Antifade with DAPI and observed under a Nikon fluorescence microscope. All experiments were performed in triplicate. The difference in organelle distribution between untreated and treated cells was calculated using the fluorescent area (normalized to total cell area using actin fluorescence) and corrected total cell fluorescence of individual cells using the image-processing ImageJ program.