Novex ecl hrp linked chemiluminiscent substrate kit

In brief, cells were seeded at a density of 4 × 10⁵ cells in a 6-well plate and incubated for 24 h. Cells were subjected to treatments as mentioned in the figure legends and processed as described earlier (Ranjan et al., 2014) [70 (link)]. The membrane was blocked with 5% non-fat milk in Tris-buffered saline for 3 h at room temperature (for phospho protein, 5% BSA was used) followed by overnight incubation with primary antibody of anti-His (1:1000), anti-cFLIP_L (1:500), anti-p53 (1:500), anti-procaspase-8 (1:500), anti-procaspasse-7 (1:500), anti-procaspase-9 (1:1000), anti-Bcl2 (1:500), anti-PARP (1:1000), anti-cytochrome C (1:500), anti-TRAF2 (1:500), anti-p65 (1:1000), anti- phospho p65 (1:1000), anti-IκBα (1:1000), anti-ubiquitin (1:1000), anti-RIP1 (1:1000), anti-cIAP2 (1:250), anti-IKKβ (1:500) anti-procaspase-1 (1:1000), anti-pro IL-1β (1:250), anti-Nlrp3 (1:1000), and anti-β-actin (1:2000) at 4 °C. Post incubation, membranes were washed and probed with HRP-conjugated secondary antibodies (1: 10,000). The membranes were incubated with a Novex^® ECL HRP-linked chemiluminiscent substrate kit as per the manufacturer’s instructions (Invitrogen) and developed in Kodak X-Omat blue film (NEN Life Sciences, Inc., Boston, MA, USA).

In brief, cells were seeded at a density of 4 × 10⁵ cells in a 6 well plate and incubated for 24 h. Cells were subjected to treatments as described in the figure legends and processed for SDS-PAGE and western blotting as described previously63 (link). In the present study, membrane was blocked with 5% non-fat milk in Tris buffered saline for 3 h at room temperature followed by overnight incubation with following primary antibodies including anti-FADD (1:1000), anti-cFLIP_L (1:500), anti- p65 (1:1000), anti-ITCH (1:500), anti-PARP (1:1000), anti-cytochrome c (1:500), anti-caspase-3 (1:1000), anti-procaspase-8 (1:200), anti-procaspasse-7 (1:500), anti-procaspase-9 (1:1000), anti-JNK1 (1:200), anti-Bcl2 (1:500), anti-Ubiquitin (1:1000), anti-RIP1 (1:1000), anti-cIAP2 (1:250), anti-IKKβ (1:500) and anti-β-actin (1:2000) at 4 °C. After washing with TBST, the membrane was probed with horseradish peroxidase conjugated secondary antibodies (1:10,000). Expression of immunoreactive proteins was detected by using Novex^® ECL HRP linked chemiluminiscent substrate kit (Invitrogen, USA) according to the instruction manual and developed in Kodak X-Omat blue film (NEN Life Sciences, Inc., Boston, MA) in the dark.