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. 2022 Apr 6;13:1880. doi: 10.1038/s41467-022-29474-3

Fig. 3. The Dectin-1a NTF is processed by SPPL2 proteases.

Fig. 3

a HEK cells stably overexpressing HA-mDectin-1a-FLAG were treated for 30 min with 40 µM (Z-LL)2-ketone (ZLL) prior to application of 50 µg/ml depleted Zymosan (dZym) for 6 h. Dectin-1a processing was visualised by western blotting using the indicated antibodies. Full-length (FL) Dectin-1 and the corresponding N-terminal fragment (NTF) are highlighted throughout the Figure. N = 2, n = 4. b BALB/c BMDCs were pre-treated for 30 min with 1 µM inhibitor X (InX) and stimulated for 6 h with 50 µg/ml dZym prior to western blot detection of Dectin-1. N = 4, n = 8. c HEK cells were transiently transfected with HA-mDectin-1a-FLAG and the indicated murine SPPL2-encoding constructs. Where indicated, cells were treated for 6 h with 50 µg/ml dZym. Dectin-1 processing was finally analysed by western blotting. The grey arrow head marks the Dectin-1a intracellular domain (ICD). N = 4, n = 4. d HeLa cells were transiently transfected with HA-mDectin-1a and mSPPL2a-myc and treated for 1 h with 50 µg/ml dZym prior to immunofluorescence analysis employing anti-HA and anti-Myc as primary antibodies. Scale bar, 10 µm. e The experiment described in d was conducted with cells transfected with mSPPL2b-myc instead of mSPPL2a-myc. Scale bar, 10 µm. N = 2, n = 2. f Expression of SPPL2a or SPPL2b was down-regulated using 20 nM of a respective siRNA mix in HEK cells stably overexpressing HA-mDectin-1a-FLAG. After stimulation with 50 µg/ml dZym for 6 h, cells were analysed by western blotting. N = 4, n = 4. g Wild type (Wt) or SPPL2a-deficient (2a KO) T-RexTM-293 cells were transfected with HA-mDectin-1a-FLAG, stimulated for 4 h with 50 µg/ml Zym and subjected to western blotting. N = 4, n = 4. h Knockout of SPPL2b in BMDC was validated by western blotting. N = 1, n = 4. i BMDC from either Wt or SPPL2b knockout (2b KO) BALB/c mice were treated for 0, 3 or 6 h with 50 µg/ml dZym. After lysis, Dectin-1 processing was visualised by western blot analysis. N = 5, n = 5. j Wt or 2b KO C57BL/6 BMDC were treated with 100 µg/ml Zym and subsequently analysed by western blotting. N = 2, n = 6. k SPPL2b-deficient BMDCs from Balb/c mice were treated with 1 µM InX for 30 min prior to stimulation with 50 µg/ml dZym for 6 h. Dectin-1 levels were evaluated by western blotting N = 3, n = 6.