Figure 4
NMIIA-P-S1943 associates with Golgi glycosyltransferases.
(a) NMIIA-P-S1943 and total NMIIA Western blot of the complexes pulled down from the lysates of EtOH-treated VA-13 cells with biotinylated control peptide, human ST3Gal1, C2GnT1, Man-II, core 3 synthase and ppGalNAcT2 N-terminal peptides, cell lysate fraction isolated by magnetic beads without peptides and cell lysate. (b) Representative 3D SIM imaging of VA-13 cells labeled with ST3Gal1 (green) and NMIIA-P-S1943 (red) before and after EtOH treatment. The Golgi area in the white box is enlarged and presented as merged channel below. (c) NMIIA-P-S1943 and ST3Gal1 Western blot of the complexes pulled down with anti-ST3Gal1 Ab from the lysate of control or EtOH-treated VA-13 cells. (d) Representative 3D SIM imaging of control rat hepatocytes and hepatocytes isolated from EtOH-fed rats and labeled with Man-II (green) and NMIIA-P-S1943 (red). The Golgi area in the white box is enlarged and presented as a merged on the right; the green channel is presented below. Colocalization area in (b,d) (white spots) indicated by white arrowheads were analyzed with the automatic threshold method of Imaris Coloc (Imaris software); bars, 10 μm. (e) Quantification of Pearson’s coefficient in cells presented in (b,d), respectively; n = 15 cells from three independent experiments; *p < 0.001. (f) NMIIA-P-S1943 and Man-II Western blot of the complexes pulled down with anti-Man-II Ab from the lysate of hepatocytes from control or EtOH-fed rats.
