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. 2012 Feb 17;7(2):e31303. doi: 10.1371/journal.pone.0031303

Figure 1. LRP1 is essential for CCN1/TNFα-, but not CHX/TNFα-induced apoptosis.

Figure 1

A. Detection of LRP1 protein levels in 15 µg of whole cell protein lysates (lanes 1, 2, 5, and 6), representing 3% of lysate used for immunoprecipitation with 7 µg/ml rabbit polyclonal anti-CCN1 antibody (lanes 3 and 4) or control IgG (lanes 7 and 8). Where indicated, 1 µM recombinant RAP was added to the medium and incubated with cells for 30 min before cell lysis. Whole cell protein lysates and immune complexes were resolved on 7.5% SDS-PAGE, immunoblotted, and 515 kDa subunit of LRP1 was detected with monoclonal anti-LRP1. B. HSFs were transfected with 80 nM non-targeting (ctrl) or LRP1 siRNA, and cell death was induced after 72 hrs by treatment of cells with CCN1 (2 µg/ml) and TNFα (10 ng/ml) for 5 hrs (left panel; *p<0.01; n = 3), or with CHX (1 µg/ml) and TNFα for 16 hrs (right panel; p = 0.13; NS-not significant, n = 3). Silencing of LRP1 was validated by immunoblot detection of the 515 kDa LRP1 subunit in control or LRP1 siRNA-treated HSFs. ERK1/2 detection serves as a loading control. C. Apoptosis in serum-starved HSFs was induced as described, in the presence or in the absence of 1 µM recombinant RAP (left panel: *p<0.01; n = 3; right panel: NS-not significant, n = 3). D. HSFs were pre-incubated with 50 µg/ml of isotype control IgG or function-blocking monoclonal antibody against LRP1, 8G1 clone (left panel: *p<0.01; n = 3; right panel: NS-not significant, n = 3).