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. 2022 Oct 14;15(2):307–325. doi: 10.1016/j.jcmgh.2022.10.005

Figure 3.

Figure 3

HDLBP promotes the MEKK1-mediated RAF1-MAPK signaling pathway.A, Immunoblotting and qRT-PCR analyses of HDLBP and RAF1 expression in the indicated HCC cells transfected with Flag-CTL or Flag-HBP. B, Immunoblots showing HDLBP and RAF1 levels in Hep3B and SNU387 cells infected with Lv-HBP or Lv-CTL. C–E, Immunoblots showing HDLBP and RAF1 levels in Huh7 and HepG2 cells transfected or infected with the indicated sequence. F, Immunoblots showing HDLBP, p-ERK, ERK, p-MEK, and MEK levels in Hep3B and SNU387 cells transfected with Flag-CTL or Flag-HBP. G, Hep3B and SNU387 cells were transfected with Flag-HBP or the empty Flag vector for 12 hours and then cotransfected with HBP-sh, HBP-res, or the corresponding empty vector for 24 hours as indicated. The protein levels of HDLBP, p-ERK, ERK, p-MEK, and MEK were measured using immunoblotting. H, Hep3B and SNU387 cells were transfected with Flag-HBP or the empty Flag vector for 24 hours and then treated with the indicated Ras inhibitor for 12 hours. The protein levels of HDLBP, p-ERK, ERK, p-MEK, and MEK were measured using immunoblotting. I, Huh7 lysates were immunoprecipitated with the anti-IgG, anti-HDLBP, or anti-RAF1 antibody and immunoblotted with the indicated antibody. J, Immunoblot s showing the levels of MEKK1, p-ERK, ERK, p-MEK, and MEK in Huh7 and HepG2 cells transfected with CTL-sh or MEKK1-sh. K, Immunoblots showing the levels of the indicated proteins in HCC cells treated with DMSO or AMG510 for 12 hours. L, Immunoblots showing the levels of the indicated proteins in Huh7 cells transfected with the indicated doses of HBP-sh1 for 12 h and then treated with AMG510 for 12 hours. M, Hep3B cells were transfected with Myc-MEKK1 or the empty Myc vector (CTL) for 12 hours and then transfected with HA-RAF1, HA-CTL, or HA fusion mutant vector for 24 hours. The levels of the indicated proteins were measured using immunoblotting. N, Hep3B cells were transfected with Flag-HBP or the empty Flag vector for 12 hours and transfected with HA-RAF or HA fusion mutant vector for 24 hours, and then transfected Huh7 cells were treated with AMG510 for 12 hours. The levels of the indicated proteins were measured using immunoblotting. O, Immunoblots showing the levels of HDLBP, and RAF1 in parental HCC cells and corresponding SR HCC cells. For all the experiments described above, the data are presented as the means ± standard deviations (SDs), and 3 independent experiments (N = 3) were performed in triplicate. ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001. ns, Not significant.