Fig 1.
Growth factor treatment does not change Golgi complex localization of GalNAc-T1 or -T2.
Serum starved HeLa cells were stimulated with 100 ng/ml of EGF for 4 h, 50 ng/ml of PDGF for 3 h or left untreated as a control. Cells were subsequently immunostained with antibodies to the Golgi complex marker TGN46 (AC’, AG’, AK’, BC’, BG’ and BK’), the ER marker calnexin (cnx) (AA’, AE’, AI’, BA’, BE’, and BI’) and either endogenous GalNAc-T1 (AB’, AF’, and AJ’) or GalNAc-T2 (BB’, BF’ and BJ’). Merged channels (AD’, AH’, AL’, BD’, BH’, and BL’) demonstrate that growth factor treatments have no effect on GalNAc-T1 or—T2 Golgi complex localization. Insets depicting a magnified view of merged imaging channels. Individual maximum projections of 30 confocal sections shown in (A) are representative of 95, 74 and 78 cells for Control, EGF and PDGF treatments, respectively, from two independent experiments. Images in (B) are representative of 73, 79, and 61 cells for Control, EGF and PDGF treatments, respectively, from two independent experiments. Scale bars, 10 μm. (C) Manders’ correlation coefficient was used to quantitate the degree of coincidence between GalNAc-T1 or—T2 with TGN46 (Golgi complex) and GalNAc-T1 or—T2 with calnexin (ER) in control, EGF and PDGF treated cells from confocal sections acquired over the entire volume of each cell. Values represent the mean ± S.D. from the number of cells described in (A) and (B).
Fig 2.
Growth factor treatment has no effect on Tn antigen localization.
Serum starved HeLa cells were either left unstimulated, treated with 100 ng/ml of EGF for 4 h or 50 ng/ml of PDGF for 3 h. Cells were subsequently immunostained with antibodies to TGN46 (AC’, AG’ and AK’), calnexin (AA’, AE’, and AI’) and the lectin HPA (AB’, AF’ and AJ’). Merged channels (AD’, AH’, and AL’) show that neither EGF nor PDGF treatment move Tn antigen away from the Golgi complex. Individual maximum projections of 30 confocal sections shown are representative of 93, 76 and 71 cells for Control, EGF and PDGF treatments, respectively, from two independent experiments. Scale bars, 10 μm. (B) To quantitate the amount of Tn antigen found in Golgi complex versus ER we calculated the Manders’ correlation coefficient for HPA/TGN46 and HPA/calnexin signals acquired over the volume of individual cells, respectively. Values represent the mean ± S.D. of quantified experimental replicates described in (A).
Fig 3.
Time course of MAPK activation following growth factor treatments.
(A) Serum starved cells were left untreated, treated with 100 ng/ml of EGF or 50 ng/ml of PDGF. At varying time points after treatment cell extracts were prepared and subjected to Western blotting with antibodies specific for P44/P42 or phosphorylated P44/P42 (P-P44/P42) as an indicator of MAPK signalling. The time course revealed that the highest level of P-P44/P42 is detected 10 minutes after treatment. Western blots shown are representative of three independent experiments. (B) Quantification of fold change in detectable P-P44/P42 as a function of time after growth factor treatment. Average fold change in P-P44/P42 is shown with error bars representing the S.D. seen from the quantification of three independent western blots from three separate growth factor treatments. (C) Quantification of amount of GalNAc-T1, -T2 and Tn antigen found in Golgi complex or ER after 10 minutes of growth factor treatments. Manders’ correlation coefficient was calculated on the data set shown in S2 and S3 Figs. Values represent the mean ± S.D.