Figure 4

Localization of KBTBD8 protein in NIH3T3 cell and after treatment with BFA or Nocodazole. NIH3T3 cells – with and without BFA or Nocodazole treatment - were fixed and co-stained with KBTBD8 antibody and Golgi matrix marker GM130 or microtubule marker protein α-Tubulin. Treatment with Brefeldin A (BFA) resulted in resorption of the Golgi membrane and Golgi marker proteins to the ER. For GM130 the expected punctuated cytoplasmic localization could be found indicating that the treatment worked. However, KBTBD8 was either no longer detectable, not co-localizing with GM130 or associated to the spindle apparatus of mitotic cells. Co-staining with α-Tubulin and Kbtbd8 in untreated cells showed no localisation of KBTBD8 at the cytoskeleton. After Nocodazole- treatment, which results in the depolarisation of microtubules and in the disperse of the Golgi stacks to the cytoplasm, GM130 and α-Tubulin showed the expected localisation whereas KBTBD8 was again either not detectable at all or no longer co-localizing with GM130. These results indicate that KBTBD8 is not a part of the Golgi matrix. Scale bar, 10 μm.