Fig. 1.

TNF-α-dependent secretion of KRS. (A) Each of 11 different human ARSs was expressed in HEK293 cells by transfection and by their expression in whole-cell lysates (WCL), and secretion into the culture media was determined by Western blotting with an anti-Myc antibody. The expected molecular masses of ARSs, in kDa, are as follows: L, 138; V, 140; A, 107; M, 101; T, 82; D, 57; G, 78; R, 75; K, 68; S, 59; HRS, 57. LRS contains ≈4 kDa extra peptide originating from the vector, KRS runs slightly bigger than the expected size in the gel for unknown reason, and VRS lacking its N-terminal 300 amino acids was used because its full-size version was not expressed well. All of the other ARSs are expressed as full length. (B) HEK293 cells transfected with human KRS were incubated, and the culture medium was harvested at the indicated times. The lack of tubulin in the culture medium indicates the absence of cell lysis resulting from physical damage. (C) The macrophage cell line, RAW264.7, and the colon cancer cell line, HCT116, were treated with TNF-α (10 ng/ml) or TGF-β (2 ng/ml), and the secretion of KRS into the culture medium was determined with its specific antibody. (D) Time course of TNF-α-induced secretion of KRS from HCT116 cells.