Figure 2.

YB-1 secretion is mediated by a calcium- and ATP-dependent non-classical secretory pathway. (a) Western blot analysis of serum-free cell culture supernatants after inhibition of classical protein secretion by monensin or brefeldin A. Representative data of at least two independent experiments are shown. Relative band intensities are indicated relative to the untreated control supernatants and uncropped Western blots available in Figure S3a. (b–e) Western blot analysis of serum-free culture supernatants after stimulation of the melanoma cells with the Ca²⁺ ionophore ionomycin and ATP (b), the combination of ionomycin and the calcium chelators BAPTA or EGTA (c), with increasing ATP concentrations (d), or the combination of ATP and the ATP complexing agent MgCl₂ (e). Relative band intensities are indicated and uncropped immunoblots presented in Figure S4. The intracellular ATP content was measured with the CellTiter-Glo^® Luminescent Cell Viability Assay (Promega) (d,e, bottom; mean ± standard deviation (SD), representative experiment of N = 2 with n = 3). Significance was determined with one-way ANOVA and subsequent Tukey’s multiple comparison test (ns = non-significant, * for p < 0.05, ** for p < 0.01, *** for p < 0.001 and **** for p < 0.0001). (f) Electron micrographs of a YB-1 specific immunogold labelling (C-terminal-antibody; red) in MelJuso cells stimulated with either ATP or ionomycin. Nucleus (N), scale bars represent 0.5 µm.