Supplementary MaterialsSupplementary Physique Legends 41419_2017_144_MOESM1_ESM. II expression were observed in plasma membranes of antigen-presenting cells derived from and gene in corresponding cell lines26. Of note, cell type specific promoter usage controlling expression of CIITA leads to the inclusion of a CARD-domain in the CIITA splice isoform 1, expressed in dendritic cells and macrophages, a structural motif also found in caspase-227. In an unbiased methodological approach, we made an attempt to expand the knowledge of caspase-2 function by means of identification of interacting factors. We found that cytosolic caspase-2 binds to the ankyrin repeat domain name of RFXANK. Although no alteration of MHC II was detected in plasma membranes of antigen-presenting cells (APC) from non-exposed caspase-2-deficient mice, an upregulation could be seen in protein lysates from gene harbors several putative in-frame start codons, the cDNA used as bait was synthesized according to the reported identification of its favored translation initiation site30. Transfection of the bait construct in yeast cells resulted in caspase-2 expression, as verified in SDS-PAGE using a specific antibody targeting the human enzyme (Fig.?1a). No processed fragments of the expressed caspase-2 construct were observed in yeast protein lysates, indicating that any prey proteins might interact with the full-length, inactive enzyme. Notably, the Y2H readout only revealed three high-confidence protein conversation hits and none of the proteins formerly reported to interact with caspase-2, such as PACAP, cyclin D3, API5/AAC11, and RAIDD2,7C9, were detected, not even among preys with low or moderate confidence in their bait conversation. Very high confidence in the conversation was, on the other hand, revealed between the caspase-2 bait and the full-length protein, as well as peptides, expressed from a total of 14 cDNA clones with complete homology to the RFXANK (regulatory factor BMS512148 inhibition X-associated ankyrin-containing protein; GenBank ID (NCBI): 523498339) splice variant 1 (“type”:”entrez-nucleotide”,”attrs”:”text”:”NM_003721.3″,”term_id”:”523498339″,”term_text”:”NM_003721.3″NM_003721.3) (Fig.?1b). The RFXANK gene is located on 19p13.11 and the corresponding transcription variant 1 translates into a 260 amino acid protein, whose most prominent signature is a proteinCprotein conversation region consisting of four ankyrin repeats31,32. Importantly, partial RFXANK cDNAs, generating truncated protein variants binding to BMS512148 inhibition caspase-2 in the Y2H screen, suggested that ankyrin repeats 1C3 or potential upstream motifs were sufficient for the conversation indicated (Fig.?1b). Open in a separate windows Fig. 1 Identification of conversation partners of caspase-2 using Y2H assaya The caspase-2 bait construct used in the screening, together with a control, was expressed in followed by analysis BMS512148 inhibition with Western blot, in order to confirm its validity. COX2 was used as a control for equal loading. The arrow indicates where cleaved caspase-2 would have appeared when separated on a gel, while using anti-caspase-2 (BD611023) for detection. b Representation of hits yielded from the Y2H screening, corresponding to the regions of hRFXANK. All hits overlapped the first three ankyrin repeats of the protein. Validation of the caspase-2-RFXANK conversation by co-immunoprecipitation?and ICC In order to validate the conversation between caspase-2 and RFXANK, as suggested by the Y2H screen, we performed co-immunoprecipitation?(co-IP) of HEK293T cell lysates after overexpression of RFXANK-myc-FLAG and a catalytically inactive caspase-2 fused to mCherry (Caspase-2C303A-mCherry). Through Rabbit Polyclonal to TAF1 immobilization of RFXANK on magnetic beads, using an RFXANK-specific antibody, BMS512148 inhibition Caspase-2C303A-mCherry could readily be detected in precipitates following co-expression. Since the antibody used was also able to capture endogenous RFXANK, a small amount of Caspase-2C303A-mCherry could be detected even without being transfected with RFXANK-myc-FLAG. Densitometry of the bands, based on the mean from three replicates of the experiment, showed that this relative density decreased in flow-through samples, compared with input (Fig.?2a). Moreover, apart from pull-down of full-length recombinant caspase-2, two processed fragments were detected in the co-IPs (Fig.?2a and Supplementary Physique?1 and 2A). These bands probably arise due to partial processing of the ectopic material. A weak signal from endogenous full-length caspase-2 was observed in the co-transfected sample (Fig.?2a). The absence of caspase-8 in immunoprecipitates support conversation specificity (Supplementary Physique?1). The same experimental setup was also carried out while using the mCherry-N1 control vector instead of.