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. 2010 Oct 4;5(10):e13158. doi: 10.1371/journal.pone.0013158

Figure 3. Western blot analysis of RNAi knockdown of Zip4 in mouse Hepa cells.

Figure 3

RNAi (two RNAi sequences) against mouse Zip4 (Z4) and a scrambled RNAi control (Con) were expressed from the U6 promoter in lentivirus vectors that also express GFP driven by the PGK promoter. Cells were infected and then purified for GFP expression by FACS. Lentivirus infected cells and uninfected Hepa cells (-) were then cultured for 24 hr in zinc-adequate medium (N) which contains normal FBS or zinc-deficient medium (CX) which contains Chelex-treated FBS. Membrane preparations from these cells were then fractionated by SDS-PAGE, transferred to a membrane, and ZIP4 was detected using an antipeptide antibody against mouse ZIP4. A prominent ∼73 kDa ZIP4 band was detected (predicted size) as well as a larger ZIP4 aggregate. ZIP1 was detected as a loading control (bottom panel). In Hepa cells a non-specific (NS) band, which is predominately cytosolic, was detected in these membrane preparations.