Fig. 4.

The effect of Surf4 knockdown on the level of apoB and apoA-I in cultured cells. A–C: Knockdown of Surf4. Huh7 and HepG2 cells were transfected with scrambled (Scram.) or one of the two Surf4 siRNAs (Surf4-1 and Surf4-2). About 36 h after transfection, the cells were incubated with oleic acid for 4 h, washed, and then incubated in DMEM without FBS and oleic acid for 16 h. Whole cell lysate was prepared. Same amount of total proteins in whole cell lysate (top) and culture medium (bottom) was applied to Western blot using antibodies indicated. Similar results were obtained from at least five experiments. The relative densitometry was the ratio of the densitometry of target to that of transferrin receptor in whole cell lysate or albumin in medium in the same condition. The ratio of the relative densitometry was the ratio of the relative densitometry of the target in Surf4 knockdown cells to that of the same target in the control cells transfected with scrambled siRNA. The relative densitometry of the target in the control cells was defined as 1. D: Coimmunoprecipitation. The same amount of whole cell lysate from Huh7 or HepG2 was incubated with the preimmune serum (Pre) or a rabbit anti-Surf4 antibody (1195Sa) and protein-G agarose. Whole cell lysate (input) and immunoprecipitated proteins (IP-Beads) were applied to Western blot with antibodies indicated. Similar results were obtained from at least three more experiments. E–G: Confocal microscopy. Huh7 cells were fixed, permeabilized, and then incubated with a goat anti-apoB (abcam) and a rabbit anti-Surf4 antibody, followed by confocal microscopy; apoB (green), Surf4 (red), and DAPI (blue). An x–y optical section of the cells illustrates the cellular distribution of proteins (magnification: 325×). Representative images were shown.