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. Author manuscript; available in PMC: 2015 Dec 1.
Published in final edited form as: Mol Cancer Res. 2014 Aug 1;12(12):1704–1716. doi: 10.1158/1541-7786.MCR-14-0291-T

Figure 5. Inhibition or knockdown of NMIIA in PC-3 and DU145 cells induces dimerization of giantin, and restores compact Golgi and C2GnT-L intra-Golgi localization.

Figure 5

(A) Colocalization of C2GnT-L and ST3Gal1 with giantin in PC-3 cells treated with DMSO, Blebbistatin or NMIIA siRNAs. (B) NMIIA W-B of the lysate of PC-3 cells after NMIIA KD. (C) Quantification of Golgi fragments (0.5 to 2 µm in size) in the cells shown in A; *, p<0.001. (D) Giantin and PDIA3 W-B of the lysates of cells after Blebbistatin treatment. (E, F) NMIIA and PDIA3 W-B of the complexes pulled down with Rab6a and giantin Abs, respectively, from the lysates of PC-3 cells treated with DMSO or Blebbistatin. (G) Quantification of Mander’s coefficient of C2GnT-L and giantin colocalization of cells presented in A; *, p<0.001. (H, I) The lysates of cells pretreated with Blebbistatin or NMIIA siRNAs were incubated with CIP with or without β-GP. (J, K) Giantin W-B of the lysates of LNCaP cells treated with scramble or Plk3 siRNAs. The control lysates were incubated with CIP with or without β-GP. (L) Images of giantin in LNCaP cells treated with scramble or Plk3 siRNAs. (M) Quantitative real-time PCR analysis of the mRNA of giantin gene in LNCaP cells treated with scramble or Plk3 siRNAs. (N) Giantin W-B of the complexes pulled down with anti-Plk3 Ab from lysates of LNCaP cells treated with scramble or Rab6a Abs, adjusted to equal amounts of giantin. (O) Images of giantin in PC-3 cells treated with scramble and Plk3 siRNAs followed by Blebbistatin, or Plk3, NMIIA and NMIIA plus Plk3 siRNAs. Bars, 10 µm. Gradient gel (4–12%) for G, H and J, and 6 or 8% gel for the rest were used.