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. 2021 Jan 28;296:100343. doi: 10.1016/j.jbc.2021.100343

Figure 2.

Figure 2

Secretion of SPINK1 inhibitor variants from transfected human embryonic kidney (HEK) 293T cells.A, secretion of untagged wildtype SPINK1 and indicated variants. B, secretion of His-tagged wildtype SPINK1 and indicated variants. Conditioned media (10 μl) were analyzed by 15% SDS-PAGE and Western blotting using an anti-SPINK1 antibody (PSKAN2) or an anti–His-tag antibody, as described in Experimental procedures section. C, trypsin inhibitory activity of conditioned media from HEK 293T cells transfected with untagged (gray bars) or His-tagged (black bars) wildtype SPINK1 and the indicated variants. SPINK1 concentrations were determined by titration against human cationic trypsin, as described in Experimental procedures section. Relative SPINK1 levels were expressed as percent of the wildtype protein. The absolute concentrations of the untagged and His-tagged wildtype proteins are also indicated (mean ± SD, n = 4). ND, not determined; levels of variant K41N could not be measured with this assay. Control media from transfections with vector only exhibited low but measurable inhibitory activity (22 ± 2 nM, n = 4), and this background was not subtracted. SPINK1, serine protease inhibitor Kazal type 1.