(A) Tn expression levels, as determined by Helix Pomatia Lectin (HPL) staining in cells expressing dominant-negative mutant Arf1 (Q71L) compared to wild-type Arf1. GRASP55 labels the Golgi apparatus. Scale bar: 10 μm. (B) Quantification of Tn expression in ERK8-depleted cells upon treatment with 50 nM GBF1 inhibitor, Golgicide, for the indicated times. (C) Quantification of Tn expression in cells following co-knockdown with ERK8 and GFP or NT5 siRNA in amounts to equivalent to double (GFP coKD), triple (GFPx2 coKD) and quadruple (GFPx3 coKD) siRNA transfection configurations. (D) SDS-PAGE analysis of protein levels of Arfs and GBF1 in single and double knockdown configurations. (E) Beta-COP (COPB) staining in control and ERK8-depleted cells. Scale bar: 10 μm. (F) Quantification of the relative numbers of COPI transport carriers (COPB) and Golgi fragments (GM130) using the granularity measurement module in MetaXpress software. At least 30 cells (non-targeting (NT) control, 37 cells; ERK8-KD, 34) were quantified for each treatment. (G) Staining of native COPI coatomer in cells treated with 5 μM Ro-31-8220 (iERK8) over the indicated times. (H) Quantification of the relative numbers of COPI transport carriers using the granularity measurement module in MetaXpress software. Twenty-five or more cells (Vehicle, 41 cells; 5 min iERK8 treatment, 25; 25 min iERK8 treatment, 57) were quantified for each treatment. (I) Staining of GalNAc-T1 (left panel) and KDEL receptor (KDELR1) localisation (right panel) in HeLa KDEL-R1-GFP stable cell line depleted of ERK8. (J) Quantification of total intensities of GalNAc-T1 and KDEL-R at the Golgi region demarcated by Giantin (Golgi protein) staining. Values on graphs indicate the mean ± SEM. **p<0.0001, *p<0.05 by two-tailed unpaired t test, relative to vehicle treated (B and H), single ERK8 knockdown (C) and NT siRNA-treated (F and J) cells. NS, not significant.