Death Associated Protein Kinase, Sigma-Aldrich, D2178

pAbmAbs reviewed Wu et al., Cell Death & Disease (2020), in which Wu evaluated Sigma-Aldrich D2178 (clone DAPK-55) against DAPK1 in Western blotting. The antibody was tested using mouse bone marrow-derived macrophages (BMDMs) and human HT-29 cells and validated using genetic validation, including WT versus Dapk1−/− BMDMs (Figure 1A) and shRNA-mediated DAPK1 knockdown in HT-29 cells (Figure 2A). In Western blotting (1:1000), the antibody detected a DAPK1 band at the expected molecular weight of appr. 160 kDa in WT BMDMs, whereas the corresponding signal was absent in Dapk1−/− BMDMs (Figure 1A). Consistent with this, the DAPK1 signal was markedly reduced in HT-29 cells following shRNA-mediated DAPK1 knockdown compared with shCtrl (Figure 2A). No meaningful off-target bands can be assessed outside the displayed molecular-weight region because only cropped Western blot images were available, and no uncropped/full-range DAPK1 blot was identified in the supplementary material. Based on the available experimental evidence and pAbmAbs review criteria, we assign 4 stars for Western blotting. The genetic validation in Figure 1A, supported by the shRNA-mediated knockdown experiment in Figure 2A, strongly supports target-specific detection of the displayed DAPK1 band, but the absence of uncropped/full-range Western blot data prevents assessment of potential off-target reactivity across the full molecular-weight range.