FIGURE 2:

RNAi-mediated knockdown of ABCG1 causes preferential loss of newly produced secretory protein and the effect reflects a deficiency in ABCG1. (A) Loss of NPY-mCherry (new protein), expressed during the last 24 h of knockdown, is significantly greater than the loss of CpepSfGFP (all protein), a portion of which was present before inducing the knockdown. Quantification from Western blots; n = 3. (B) Comparable loss of CpepSfGFP when siRNA targeted to the 3′-UTR of ABCG1 is substituted for the siRNA smart pool. Quantification from Western blots; n = 3. (C) Fluorescence images showing extensive (but not full) colocalization of stably expressed N-terminally tagged GFP-ABCG1 and of the nonfunctional Walker domain mutant GFP-ABCG1(K124M) with concentrated proinsulin and the trans-Golgi marker Golgin97. (D) Loss of NPY-mCherry caused by siRNA targeted to the 3′-UTR of ABCG1 occurs in control INS1 cells; n = 6. Loss is averted in INS1 cells stably expressing a low level of GFP-ABCG1 chimera lacking the 3′-UTR but not when the cells express nonfunctional GFP-ABCG1(K124M). Quantification from Western blots; n = 3. Data are presented as means ± SEM. p values are determined by Student’s t test; *, p < 0.05; **, p < 0.01; ****, p < 0.0001.