Figure 1: USP17 expression is required for chemotaxis.

Human PBMCs (a,b) or HeLa cells (c,d) were stimulated with CXCL12 (100 ng ml⁻¹) for the indicated times. USP17 mRNA expression was assessed by RT–PCR (a,c) and USP17 protein was determined by immunoprecipitation (IP), followed by immunoblotting (IB) (b,d). In mRNA images, β-actin was used as a loading control, whereas γ-tubulin was the control for western blotting. (e) Jurkat T cells were serum starved and then stimulated with CXCL12 (100 ng ml⁻¹) for 0, 5, 10 and 15 min. USP17 mRNA was analysed by RT–PCR. glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was also analysed as a loading control. Human neutrophils extracted from blood (f) or the monocytic cell line U937 (g) were stimulated with CXCL8 (100 ng ml⁻¹) for the indicated times. USP17 protein was immunoprecipitated then blotted for USP17. Whole-cell lysates were analysed for γ-tubulin as a loading control. (h,i) HeLa cells were transfected with either scrambled shRNA, USP17 shRNA1 or CYLD shRNA (h) or scrambled and two additional shRNAs targeting USP17 (shRNA2 and shRNA3) (i). Cells were left unstimulated (grey bars) or stimulated with CXCL12 (20 ng ml⁻¹) (black bars) and chemotaxis was measured in modified Boyden chambers. RT–PCR was used to determine USP17 mRNA expression and β-actin was used as a loading control. Experiments were conducted in triplicate; values are mean±s.e.m.; and statistical analyses are relative to untreated control by one-way analysis of variance. **P>0.05.