Human BAK Antibody

Detection of BAK by Western Blot

Role of TRAF3 & pro-apoptotic p38 & JNK kinases in mCD40L-mediated intrinsic & extrinsic cell death signalling pathways. (c) Untreated (‘C’) & mCD40L-treated (‘mL’) HCT116 & SW480-CD40 in the presence of 5µM JNK inhibitor SP600125 or p38 inhibitor SB202190 used to detect phosphorylated JNK (p-JNK) & p38 (p-p38), pro-apoptotic Bak or Bax, & TRAIL protein by immunoblotting at 3 h & 6 h post CD40 ligation. Lysates from HCT116 & SW480-CD40 cell cultures treated with mCD40L for 6 h in the absence of inhibitor included (denoted as positive control, ‘PC’) per experiment, respectively. Equal loading for human epithelial cell lysate was confirmed by CK18 detection in all experiments. Image collected & cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36291141), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of BAK by Western Blot

Role of TRAF3 & pro-apoptotic p38 & JNK kinases in mCD40L-mediated intrinsic & extrinsic cell death signalling pathways. (c) Untreated (‘C’) & mCD40L-treated (‘mL’) HCT116 & SW480-CD40 in the presence of 5µM JNK inhibitor SP600125 or p38 inhibitor SB202190 used to detect phosphorylated JNK (p-JNK) & p38 (p-p38), pro-apoptotic Bak or Bax, & TRAIL protein by immunoblotting at 3 h & 6 h post CD40 ligation. Lysates from HCT116 & SW480-CD40 cell cultures treated with mCD40L for 6 h in the absence of inhibitor included (denoted as positive control, ‘PC’) per experiment, respectively. Equal loading for human epithelial cell lysate was confirmed by CK18 detection in all experiments. Image collected & cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36291141), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of BAK by Western Blot

Rapid induction of the mitochondrial (intrinsic) apoptotic pathway and role of caspase activation in cell death. (a) Expression of Bax and Bak proteins was detected in controls (‘C’) versus mCD40L-treated (‘mL’) HCT116 and SW480-CD40 cells by immunoblotting at the indicated time points. Equal loading for human epithelial cell lysate was confirmed by CK18 detection. (b) Control (‘C’) and mCD40L-treated (‘mL’) HCT116 and SW480-CD40 cells were used to prepare cytoplasmic (‘Cyto’) and mitochondrial (‘Mito’) sub-cellular fractions for the detection of cytochrome c (Cyto c) protein by immunoblotting at 6 h post CD40 ligation. Detection of Bcl-2 and GAPDH proteins was employed to confirm sub-cellular fractionation (mitochondrial and cytoplasmic, respectively). (c) HCT116 cells were treated with mCD40L in the absence (vehicle control–denoted ‘Control’) or presence of 100 µM inhibitor of caspase-8 (z-IETD-FMK), caspase-9 (z-LEHD-FMK), caspase-10 (z-AEVD-FMK) or pan-caspase inhibitor (z-VAD-FMK). Cell death was detected 24 h later using the CytoTox-Glo assay. Results are presented as Cell death Fold increase in background-corrected RLU readings relative to control (mCD40L treatment versus controls) and are representative of 3 independent experiments. Bars show mean fold change (comparing caspase inhibitor-treated versus vehicle control cultures) for 5–6 technical replicates ± SEM. NS denotes non-significance (p > 0.05) and *** p < 0.001. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36291141), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of BAK by Western Blot

Rapid induction of the mitochondrial (intrinsic) apoptotic pathway and role of caspase activation in cell death. (a) Expression of Bax and Bak proteins was detected in controls (‘C’) versus mCD40L-treated (‘mL’) HCT116 and SW480-CD40 cells by immunoblotting at the indicated time points. Equal loading for human epithelial cell lysate was confirmed by CK18 detection. (b) Control (‘C’) and mCD40L-treated (‘mL’) HCT116 and SW480-CD40 cells were used to prepare cytoplasmic (‘Cyto’) and mitochondrial (‘Mito’) sub-cellular fractions for the detection of cytochrome c (Cyto c) protein by immunoblotting at 6 h post CD40 ligation. Detection of Bcl-2 and GAPDH proteins was employed to confirm sub-cellular fractionation (mitochondrial and cytoplasmic, respectively). (c) HCT116 cells were treated with mCD40L in the absence (vehicle control–denoted ‘Control’) or presence of 100 µM inhibitor of caspase-8 (z-IETD-FMK), caspase-9 (z-LEHD-FMK), caspase-10 (z-AEVD-FMK) or pan-caspase inhibitor (z-VAD-FMK). Cell death was detected 24 h later using the CytoTox-Glo assay. Results are presented as Cell death Fold increase in background-corrected RLU readings relative to control (mCD40L treatment versus controls) and are representative of 3 independent experiments. Bars show mean fold change (comparing caspase inhibitor-treated versus vehicle control cultures) for 5–6 technical replicates ± SEM. NS denotes non-significance (p > 0.05) and *** p < 0.001. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36291141), licensed under a CC-BY license. Not internally tested by R&D Systems.