Figure 1.

Sec23A is required for in vitro activation of primary rat HSCs. (a and b) Primary rat HSCs isolated by in situ perfusion were cultured for 1 to 10 days. Cells were collected for RNA extraction, followed by qRT-PCR (n = 5). (c and d) Primary rat HSCs isolated by in situ perfusion were cultured for 1, 6 and 10 days. Proteins were extracted and analysed by SDS-PAGE, followed by western blotting with anti-Sec23A, anti-α-SMA, anti-cTAGE5, anti-Sec12 and anti-β-actin antibodies. (c) Representative immunoblots. (d) Quantification of immunoblots (n = 3). The band intensities were normalized to those of β-actin. (e) Primary rat HSCs isolated by in situ perfusion were cultured for 2 to 6 days. Cells were fixed and stained with anti-Sec23A and α-SMA antibodies, and DAPI. Bars, 25 μm. (f and g) Primary rat HSCs isolated by in situ perfusion were cultured for 2 days and transfected with the indicated siRNA(s). After 4 days, whole cell proteins were extracted and analysed by SDS-PAGE, followed by western blotting with anti-Sec23A, anti-α-SMA and anti-β-actin antibodies. Medium was collected and the proteins were precipitated for SDS-PAGE, followed by western blotting with anti-collagen I antibody. (f) Representative immunoblots. (g) Quantification of immunoblots (n = 3). The band intensities were normalized to those of β-actin. (h) Primary rat HSCs isolated by in situ perfusion were cultured for 2 days and transfected with the indicated siRNA(s). After 4 days, cells were fixed and stained with anti-Sec23A and anti-α-SMA antibodies. Bars, 50 μm. (i) Primary rat HSCs isolated by in situ perfusion were cultured for 1 to 10 days. Cells were collected for RNA extraction and qRT-PCR (n = 5). Error bars represent mean ± SEM. *P < 0.05. Uncropped original blots were shown in Fig. S3.