Stable expression and visualization of Mat-8 (FXYD-3) tagged with a fluorescent protein in Chinese Hamster Ovary (CHO)-K1 cells

Abstract

A type I transmembrane protein, Mat-8 (FXYD-3), was tagged with fluorescent protein, Discosoma red fluorescent protein, at the carboxyl terminal cytoplasmic tail, and stably expressed in Chinese Hamster Ovary (CHO)-K1 cells. The fluorescence signal was distributed in intracellular membranes, being not only detected around the nuclear envelope but also partly overlapping with an endoplasmic reticulum marker. Subcellular fractionation by density gradient centrifugation supported this partial overlapping. The spherical structures observed were not colocalized with markers for lysosomes, endosomes, and Golgi bodies, suggesting that Mat-8 is distributed in a distinct endoplasmic reticulum region and the nuclear envelope after synthesis on membrane-bound ribosomes.

Intracellular Localization of ABC Transporter TAPL Differs between Transient and Stable Expression

“…CHO-K1 cells were cultured in Ham's F-12 medium (GIBCO) supplemented with 7% (v/v) fetal bovine serum (JRH Biosciences) [13]. Transient transfection was carried out as follows: on a Lab-tek II 4-well chamber slide (NalgenNunc), 5 × 10 3 cells were seeded in 1 ml of the medium and then cultured overnight.…”

“…Cells were cultured further for 24 hr and subjected to analysis by immunofluorescence microscopy. Stable transformants were isolated in the presence of 0.1 mg/ml G418 (Nacalai) [13].…”

“…The cells were fixed in methanol and then subjected to antibody-treatment as described previously [13] [14]. The antibodies [protein disulfide isomerase (PDI), cathepsin D and mannose-6-phosphate receptor (M6PR)] were diluted with 0.2% (w/v) gelatin in PBS, and then an aliquot (0.2 ml/well) was reacted at room temperature for 1 hr, followed by 5 min incubation with a gelatin solution (0.5 ml/well) three times.…”

Down-Regulation of FXYD3 Expression in Human Lung Cancers

“…Mammary tumor 8 kDa (MAT8)/FXDY domain-containing ion transport regulator 3 (FXYD3), a FXYD-containing Na,K-ATPase ion channel regulator, was first identified as a protein highly expressed in murine breast tumor initiated by oncogenic ras (v-Ha-ras) or neu (her2, egfr2) [1][2][3][4][5][6][7][8][9][10][11][12][13] and later demonstrated to occur at (inner-) cellular membranes and to be involved in the regulation of chloride conductance. [11][12][13] Recent studies found FXYD3 to be overexpressed breast, prostate, and pancreatic cancers, 5,14,15 and suggested that it promotes carcinogenesis by providing a growth advantage.…”

“…[11][12][13] Recent studies found FXYD3 to be overexpressed breast, prostate, and pancreatic cancers, 5,14,15 and suggested that it promotes carcinogenesis by providing a growth advantage. 14,15 Moreover, its expression was reported to be up-regulated in response to stimuli inducing cellular differentiation 16 and by treatment with an anticancer drug, 5-fluofouracil, in colon and/or breast cancer cells.…”