Figure S1. Characterization of anti-XK... | Rockefeller University Press

Figure S1.

$Characterization of anti-XK antibodies and the XK-EGFP reporter. (A) Western blot analysis of lysates of cells transfected with the indicated amounts of plasmids expressing WT XK. (B) Densitometry of blot analyses in A showed that signals for the protein band of ∼43 kD identified with an arrow increased in a plasmid dose-dependent manner. Signals for the protein band of ∼52 kD indicated by an open arrowhead were very weak at basal state, but also appeared to increase in a plasmid dose-dependent manner. Signals for the protein bands of 90 and 100 kD marked by a star symbol remained constant regardless of the amount of plasmids transfected, suggesting that they are crossreactive. (C) Western blot analysis of cytosol and total membranes prepared from STHdhQ7/Q7 cells. The protein bands of 43 and 52 kD, respectively, were present in total membranes, whereas the protein bands of 90 and 100 kD were cytosolic, further supporting their crossreactivity to XK antibodies. (D) Schematic representation of the XK-EGFP reporter. (E) STHdhQ7/Q7 cells were transfected with pcDNA3.1-XK for 16 h and further cultured in the presence or absence of β-cycloheximide (CYX) for 5 h. Cells were collected for preparing homogenates by passing through a 25-gauge needle. Postnuclear supernatants were overlaid on a discontinuous Nycodenz gradient and centrifuged as in Materials and methods. The same volume of each fraction was analyzed by Western blot. Protein bands identified by fixed arrowheads were detected by other antibodies left over in the XK antibody solutions which were reused. XK-EGFP was expressed at the expected size. The open arrowhead indicated the 52 kD isoform of XK. Source data are available for this figure: SourceData FS1.$

Characterization of anti-XK antibodies and the XK-EGFP reporter. (A) Western blot analysis of lysates of cells transfected with the indicated amounts of plasmids expressing WT XK. (B) Densitometry of blot analyses in A showed that signals for the protein band of ∼43 kD identified with an arrow increased in a plasmid dose-dependent manner. Signals for the protein band of ∼52 kD indicated by an open arrowhead were very weak at basal state, but also appeared to increase in a plasmid dose-dependent manner. Signals for the protein bands of 90 and 100 kD marked by a star symbol remained constant regardless of the amount of plasmids transfected, suggesting that they are crossreactive. (C) Western blot analysis of cytosol and total membranes prepared from STHdhQ7/Q7 cells. The protein bands of 43 and 52 kD, respectively, were present in total membranes, whereas the protein bands of 90 and 100 kD were cytosolic, further supporting their crossreactivity to XK antibodies. (D) Schematic representation of the XK-EGFP reporter. (E) STHdhQ7/Q7 cells were transfected with pcDNA3.1-XK for 16 h and further cultured in the presence or absence of β-cycloheximide (CYX) for 5 h. Cells were collected for preparing homogenates by passing through a 25-gauge needle. Postnuclear supernatants were overlaid on a discontinuous Nycodenz gradient and centrifuged as in Materials and methods. The same volume of each fraction was analyzed by Western blot. Protein bands identified by fixed arrowheads were detected by other antibodies left over in the XK antibody solutions which were reused. XK-EGFP was expressed at the expected size. The open arrowhead indicated the 52 kD isoform of XK. Source data are available for this figure: SourceData FS1.

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