Biochemical and Biophysical Research Communications
Subcellular localization and N-glycosylation of human ABCC6, expressed in MDCKII cells
Section snippets
Materials and methods
Materials. Tunicamycin, trypsin (type XIII), and soybean trypsin inhibitor (type 1-S) were from Sigma, N-Glycosydase F from Boehringer–Mannheim GmbH. The polyclonal antibody K14 was a kind gift from Stieger, see [21]. The chicken polyclonal anti-Na,K-ATPase antibody was obtained from Chemicon International (Temecula, CA), the Alexa Fluor 488 goat anti-rat IgG (H + L), and the Alexa Fluor 594 goat anti-chicken IgG (H + L) were obtained from Molecular Probes (Eugene, USA).
Expression of human ABCC6 in
Expression and localization of human ABCC6 in polarized cells
In order to provide expression of human ABCC6 in a polarized monolayer, a retroviral transduction system and kidney-derived MDCKII cells have been utilized. Cell lines with various expression levels were produced by growing individual cell-derived ABCC6-transduced cells. By using this method, we obtained stable MDCKII clones with high yield of ABCC6 expression.
For the detection of the subcellular localization of human ABCC6, the polarized cultures of transduced MDCKII cells were immunostained
Acknowledgements
The technical help by Györgyi Demeter, Judit Kis, and Ilona Zombori is gratefully acknowledged. Thanks are due to Dr. Spat of the Department of Physiology, Semmelweis University of Budapest for making the confocal microscope available for the present study. K14 polyclonal antibody was a kind gift of Dr. B. Stieger. This work has been supported by grants from OTKA (T31952, T35926, T38337, and F 034247) by FKFP 0130/2001 and by NKFP 1/047 of Hungary. A. Váradi and B. Sarkadi are awardees of the
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Membrane insertion and topology of the amino-terminal domain TMD0 of multidrug-resistance associated protein 6 (MRP6)
2015, FEBS LettersCitation Excerpt :Taken together the results demonstrate that TMD0 consists of five TMs and its N- and C-termini are on the external and cytosolic sides, respectively (Fig. 1A), which is consistent with the TMHMM prediction. The finding that the N-terminus is on the lumenal side of the ER membrane and that N15 is glycosylated is supported by previous observations with MRP6 expressed in MDCKII cells [2] and with the first 102 residues of MRP6 expressed in vitro [45] and in HEK-293T cells [46]. In the latter report it was also demonstrated that the loop following TM2 is localized in the ER lumen.
Analysis of pseudoxanthoma elasticum-causing missense mutants of ABCC6 in vivo; Pharmacological correction of the mislocalized proteins
2014, Journal of Investigative DermatologyCitation Excerpt :The transfection was performed using the calcium phosphate method. Cell clones overexpressing ABCC6 were selected by end-point dilution (Sinkó et al., 2003). For immunocytochemistry, MDCKII cells were first washed and fixed with 4% paraformaldehyde and precooled methanol, and they were then incubated with blocking buffer for 1 hour at room temperature.
The hepatitis B x antigen anti-apoptotic effector URG7 is localized to the endoplasmic reticulum membrane
2013, FEBS LettersCitation Excerpt :Another difference is that URG7, unlike MRP6, is most likely targeted post-translationally to the ER membrane because its C-tail contains only 43 residues, which is just about enough to span the ribosome tunnel [18,19]. Although MRP6 and URG7 share targeting signals for the ER, MRP6 has been suggested to be localized to the plasma membrane or mitochondria-associated membranes [6,7]. Our results therefore further suggest that the ER retention signal for URG7 is found among residues 74–99 that are different from those of MRP6.
ABCC6 does not transport vitamin K3-glutathione conjugate from the liver: Relevance to pathomechanisms of pseudoxanthoma elasticum
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