Fig. 2.

Effects of ATOX1, CCS, and ATOX1/CCS knockdown on copper uptake. A: Western blot analysis of ATOX1, CCS, and actin loading controls from lysates of replicate wells used in ⁶⁴Cu uptakes (50 μg/lane) and surface-biotinylated hCTR1 and Na-K-ATPase (α-subunit) controls from the replicate wells. Primary antibodies (ab) are shown at right. B: ⁶⁴Cu uptake in siRNA-treated cells. Tet-induced HEK293 cells overexpressing (FLAG-tagged) hCTR1 were transfected with siRNA oligos targeting: ATOX1, CCS, ATOX1, and CCS or control siRNA for 72 h before ⁶⁴Cu uptake assays (see experimental procedures). In this and each subsequent figure, the means ± SD of a single experiment from triplicate wells are shown and are representative of those from 3–5 separate experiments. C: ⁶⁴Cu uptake normalized to the amount of hCTR1 in the membrane relative to the control siRNA cells (control set at 100%). Average of 3 experiments shown in B. D: copper chaperone knockdown experiments done exactly as in B, without tet-induced overexpression of hCTR1. *P < 0.05, significantly different value vs. control by Student's t-test, unpaired.