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. 2021 Sep 8;297(4):101177. doi: 10.1016/j.jbc.2021.101177

Figure 5.

Figure 5

ASGR1 regulates LDLR levels and functionality independently of PCSK9. HepG2-naïve cells (A, C and D) or HepG2-PCSK9-KO cells (B and E) were transfected for 48 h with nontargeting siRNA (siCnt) or siRNA ASGR1 (siASGR1) at final concentrations of 20 nM. Cells were then incubated for the last 20 h with media only (−PCSK9; SFM) or media containing purified PCSK9 (3 μg/ml) (+PCSK9) (A–C). A and B, total protein levels of LDLR and ASGR1 were assessed by Western blot. In (A) the Western blot of the siCnt condition was reused from Figure 2B. C, immunofluorescence microscopy of LDLR (green signal) and ASGR1 (white signal) at the plasma membrane. D and E, immunofluorescence microscopy of plasma membrane LDLR (green signal) and (E) plasma membrane ASGR1 (white signal). LDLR functionality was assessed by DiI-LDL (5 μg/ml) internalization for 2 h at 37 °C before fixation (red signal). The scale bar represents 15 μm. F, quantitative RT-PCR analysis was performed on HepG2-naïve cells transfected for 48 h with nontargeting siRNA (siCnt) or siRNA ASGR1 (siASGR1) at final concentrations of 20 nM. Data are representative of at least three independent experiments. Quantifications are averages ±SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 (t test).