Fig. 2: HSPA8 targets RIP3 to prevent the execution of necroptosis.

a HSPA8 blocked RIP3 oligomerization-induced necroptosis. After 36 h of transfection of siHSPA8 oligos into the mRIP3-2×FKBP-3T3 cells, RIP3 oligomerization was induced by adding 20 nM FKBP dimerizer (AP20187, C₈₂H₁₀₇N₅O₂₀). The dimerizer competitor Tac (10 μM) was used as a negative control to block RIP3 oligomerization-induced necroptosis. The HSPA8 knockdown efficiency was tested by immunoblotting (right panel). b Schematic representation of the full-length and truncation variants of RIP3. FL, full-length; NT, N-terminal; CT, C-terminal; KD, kinase domain; ID, intermediate domain; RHIM, RIP homotypic interaction motif. c, d Mapping the HSPA8 binding region of RIP3 by co-immunoprecipitation. The Flag-tagged truncated RIP3 (as shown in b) and Myc-tagged HSPA8 cDNAs were co-transfected into 293FT cells. After 24 h of transfection, the whole-cell lysates were immunoprecipitated with anti-Myc beads, and immunoblotted with the indicated antibodies. Results are reported from one representative experiment from at least three independent repeats. e Mutations of the RHIM-core of RIP3 did not disrupt the interaction with HSPA8. Flag-tagged WT or the RHIM-core mutant, RIP3-4A mutant (VQVG into AAAA), was co-transfected with Myc-tagged HSPA8 into 293FT cells. Immunoprecipitation with anti-Myc beads was carried out as described in c and d. f In vitro His pull-down assay verified the interaction between HSPA8 and RIP3-RHIM. GST-tagged HSPA8-SBD domain (GST-HSPA8-SBD_385–647) and His-tagged RIP3-RHIM region with the mutation of the RHIM-core (His-Sumo-RIP3_388–518-4A) were purified from E. coli separately and mixed at the ratio of 1:1 (50 μM each). The His-tagged RIP3-RHIM was used as bait; the GST-tagged HSAP8 was used as prey and was pulled down by His-tagged RIP3-RHIM. g Co-immunoprecipitation of endogenous RIP3 and HSPA8. The L929 cells were transfected with the indicated siRNAs. After 48 h, the whole-cell lysate was incubated with anti-RIP3 antibody and protein A/G beads. The immunoprecipitated complexes were probed with the indicated antibodies. P values were determined by unpaired two-tailed Student’s t-test with Welch’s correction. **P < 0.01; ***P < 0.005. All results are reported from one representative experiment from at least three independent repeats.