Fig. 10.

TXNL1 acts as a general chaperone as it prevents protein aggregation in heat-treated cell lysates. Cell lysates were prepared from HEK293T, A549 and AGS cells and first evaluated for (A) endogenous levels of TXNL1, Trx1, TrxR1, and HSP27 using Western blots, with β-actin used as loading control. (B) Protein of whole cell lysates (1 mg/ml) of the corresponding cell lines (indicated at the top of the figure) were incubated at 4 °C or 45 °C for 90 min in PBS containing 1 mM DTT. After incubation, soluble (SF) and insoluble fractions (ISF) were separated by centrifugation and analyzed by SDS-PAGE followed by Coomassie staining. Samples of 293T cell lysate were subsequently incubated at 45 °C for 90 min with addition of varying concentrations (0–10 μM) of (C) HSP27, (D) Trx1, or (E) TXNL1, before they were analyzed as in (B). Lysates from (F) A549 or G) AGS cells were also incubated with either HSP27 (4 μM), Trx1 (10 μM) or TXNL1 (10 μM) for 90 min at 45 °C and analyzed as in (B). Positions and size of molecular weight markers are shown in kDa and bands corresponding to the respective recombinant proteins are indicated with arrows and labels. Total amounts of proteins in lanes containing the soluble (SF) and insoluble fractions (ISF) were quantified using ImageJ software. For each experiment (C–G), raw values were normalized to the total amount of protein (soluble and insoluble), set to 100% and the bar graphs visualize the proportions of SF vs. ISF, with values and error bars representing mean ± S.D. from three independent experiments.