Figure 7
Hevin contains N-linked glycosylated isoforms, but expression is not regulated by loss of SPARC expression.
(a) PS-1 cell lysates were treated with PNGase-F glycosidase and analysed by western blot using antibodies to Hevin and β actin as a loading control. Images are representative of 3 independent replicates, and the mean molecular weight observed for each band is indicated next to the blots. Arrow indicates de-glycosylated band. (b–e) PS-1 stellate cells were treated with anti-SPARC (+) or control (−) siRNA for 48 hours. Cell lysates were then analysed by western blot using antibodies to SPARC to confirm knockdown (b), Hevin N-terminus (c), or Hevin C-terminus (d). On average 90% knockdown of SPARC expression was achieved in the three experiments performed (+/− 6%). In (e), the graph shows mean signal intensity +/− SEM for the Hevin N-terminal antibody, standardised to β actin and relative to the control, for cells treated with SPARC (KD) or control siRNA. Images are representative of 3 independent replicates, and statistical significance was measured using the Student’s t-test (unpaired, two-tailed). p-values are indicated in the graph. Uncropped blots are shown in Suppl. Figure 4.
