Fig. 5

TRAP isoform prevalence after CtsK inhibition. a Cells were treated with 100 nM CtsK inhibitor (MK-0822, Odanacatib) and lysates and conditioned media were collected either directly after 24 h treatment (AT) or after an additional recovery period for 24 h after the treatment (R) at 48 h. Controls were treated with equal concentration of DMSO. b One representative Western blot of TRAP isoform expression and processing in mock control and TRAP3^high cells is shown. Membranes were stained with anti-total TRAP antibody and anti-β-Actin antibody in lysates. Quantification of TRAP isoforms in mock control and TRAP3^high cells c TRAP 5a 35 kDa in lysates, d TRAP 5a 35 kDa in conditioned media and TRAP 5b N-terminal subunits ~ 23 kDa) in mock control and TRAP3^high cell lysate. Western blots were normalized to β-Actin (independent experiments n = 3, bar graphs represent means and Standard error). Expression levels were normalized to TRAP 5a expression in the mock control cells and statistically compared by ANOVA test (Fisher’s LSD test). f Colocalization of total TRAP and proCtsK was measured in TRAP3^high cells in presence of MK-0822. Means (independent experiments n = 3, technical replicates 8–20 cells in at least 3 fields of view) from independent cultures were statistically compared by t- test. g Compartmentalization of total TRAP-signal, means from independent experiments of cells treated with MK-0822 were statistically compared by one-way ANOVA with Tukey’s correction for multiple comparisons. * p < 0.05, ** p < 0.01 *** p < 0.001