Figure 4. Lrp1-AS reverses the Hmgb2-mediated increase in Srebp1-dependent Lrp1 expression.

(A) Lrp1 and Lrp1-AS levels in RAW264.7 cells transfected with control or Srebp1 siRNA. The control siRNA has a sequence with no homology to any gene. Western bloting (WB) confirms Srebp1 silencing (upper).

(B) Lrp1 levels after overexpression of mature Srebp1a-Flag and/or Hmgb1-HA/2-Myc in RAW264.7 cells (upper). Expression of exogenous proteins was monitored by WB with the indicated antibodies (lower). An antibody to β-actin was used as loading control in (A) and (B).

(C) Luciferase reporter assay after co-transfection of the Lrp1 promoter-coupled luciferase construct (upper, pGK3-Lrp1 pro.) together with overexpression of Srebp1-Flag and Hmgb1-HA/2-Myc in RAW264.7 cells. Firefly luciferase activity was normalized to Renilla luciferase activity.

(D, E) Reciprocal immunoprecipitation (IP) of endogenous Srebp1 or Hmgb1/2 from RAW264.7 cells followed by WB with the indicated antibodies. Rabbit IgG was used as negative control.

(F) Lrp1 levels after overexpression of mature Srebp1a-Flag and Hmgb2-Myc with or without Lrp1-AS RNA in RAW264.7 cells.

(G) Luciferase activity after co-transfection of the Lrp1 promoter-coupled luciferase construct together with overexpression of mature Srebp1-Flag and Hmgb2-Myc with or without Lrp1-AS RNA in RAW264.7 cells. Firefly luciferase activity was normalized to Renilla luciferase activity.

(H) Quantitative chromatin immunoprecipitation (qChIP) analysis with Srebp1 antibody or control IgG from RAW264.7 cells transfected with control or Lrp1-AS siRNA (lower). Diagram of the Lrp1 promoter, showing the two tandem SREBP Responsive Elements-like motifs (SRE-likes in upper) (Bist et al., 1997). Mean ± s.d. (n = 3 replicates) are shown in all bar graphs.*P < 0.05, **P < 0.01, ***P < 0.001 determined by ANOVA.