We hypothesised that unlike rhTRAIL, agonistic DR4/5 antibodies do not trigger receptor internalisation resulting in JNK activation in a different cellular compartment. Navitoclax Sigma Colo205 cells were treated with FITC-labelled rhTRAIL or agonistic DR4/5 antibodies cross-linked by a FITC-labelled secondary antibody for 30min at either 37��C or +4��C and their internalisation analysed by flow cytometry. An acid wash step was carried out at +4��C after the incubation to remove all non-internalised, surface bound ligand/antibody ensuring that any fluorescent signal was due to internalised ligand/antibody-receptor complexes. The flow cytometric analysis showed that both rhTRAIL and agonistic antibodies bound to the TRAIL receptors at both 37��C and +4��C and were all internalised to a similar extent, when incubated at 37��C but not at +4��C (Figure 4A and B).

The same samples were also tested for the ability of rhTRAIL and agonistic antibodies to bind to and activate their receptors in these conditions. At the end of the 30min incubation, unbound rhTRAIL or agonistic antibodies were removed by a wash step. The samples were incubated for an additional 3h and induction of apoptosis was detected as a measure of receptor activation. The extent of apoptosis was the same regardless of incubation temperature (Figure 4C), confirming that all treatment conditions enabled ligand/antibody-receptor interaction. These results argue against the compartmentalisation hypothesis. Figure 4 Receptor internalisation induced by rhTRAIL and agonistic DR4/5 antibodies.

(A) Flow cytometric analysis of ligated receptor internalisation in Colo205 cells. Cells were treated with DR4/5 agonistic antibodies (5nM) cross-linked with FITC-labelled … rhTRAIL and agonistic DR4/5 antibodies phosphorylate distinct JNK1 isoforms To address the different effects of JNK inhibition on apoptosis, we next investigated which JNK isoforms were phosphorylated by rhTRAIL and the DR4/5 antibodies. JNK1 was immunoprecipitated from rhTRAIL-treated and agonistic antibody-treated Dacomitinib Colo205 and HCT15 cells. Immunoprecipitates were electrophoresed on SDS�CPAGE and probed for phosphorylated JNK, to identify which JNK1 isoforms were activated by the different treatments. For quantification, blots were also probed for total JNK1 (Figure 5A) and the densitometric ratio of p-JNK1-long or -short to total JNK1-long or -short was calculated (Figure 5B).