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. 2017 Jan 23;7:41173. doi: 10.1038/srep41173

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Copyright © 2017, The Author(s)

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(a) Expression level of transduced proteins. HLFs were transduced with adenovirus expressing S348A/S409A for 48 h (lane 1) or wt HA-LARP6 for 4 h (lane 2) or 48 h (lane 3) and analyzed by Western blot using anti-LARP6 antibody. Lane 4; expression of endogenous LARP6. β-actin (ACT): loading control. (b) Dominant negative effect in HLFs. HLFs were transduced with wt HA-LARP6 or S348A/S409A mutant and collagen polypeptides (COL1A1 and COL1A2) were analyzed intracellularly (CELL) and in the medium (MED). FIB: fibronectin loading control. (c) Expression of collagen mRNAs. Total RNA from cells in (b) was analyzed for expression of COL1A1 and COL1A2 mRNAs by real-time PCR. Relative expression normalized to β-actin mRNA is shown as mean ± SD (n = 3). (d) Secretion rate of collagen from HLFs expressing S348A/S409A. Wt HA-LARP6 and S348A/S409A mutant were expressed in HLFs and accumulation of collagen polypeptides in the medium was analyzed at indicated time points. Loading control: fibronectin (FIB). (e) Collagen polypeptides are not degraded in the cellular medium. Cellular medium from the 3 h time point in (d) was incubated for additional 6 h at 37 °C and analyzed by Western blot. (f) S348A/S409A mutant does not change intracellular level of collagen. Cells in (d) were analyzed for intracellular collagen level. (g) Hyper-modifications of COL1A2 polypeptide in cells expressing S348A/S409A. The pI of COL1A2 polypeptide in cells expressing wt LARP6 and S348A/S409A was analyzed by 2DGE. Arrow: pI of COL1A2 in control cells. (h) S451D mutant confers resistance to rapamycin. HLFs were transduced with wt HA-LARP6 or S451D mutant, treated with (RAPA) or without (CON) rapamycin and collagen polypeptides (COL1A1 and COL1A2) were analyzed intracellularly (CELL) and in the medium (MED). Fibronectin (FIB) and actin (ACT) were used as loading control. HA-LARP6, expression of transfected proteins.