Fig. 4

C101A LPL and C42A LPL retain their function under pro-oxidative conditions. Actin-bundling capacity of recombinant wt, C101A and C42A LPL under pro-oxidative conditions. Recombinant LPL pretreated with the indicated concentrations of H₂O₂ was coincubated with G-actin for 4 h. Actin bundles were separated from F-actin and G-actin by low-speed centrifugation. The pellet and supernatant fractions were loaded onto SDS-polyacrylamide gels and stained with Coomassie Brilliant Blue. a Representative Coomassie-stained gel for recombinant wt LPL. P, pellet; S, supernatant (n = 5). b, c Percent quantification of b actin and c LPL in the pellet fraction. The percentage of protein in the pellet was calculated by taking the ratio of the corresponding signal in the pellet fraction to the total intensity (of supernatant and pellet) (n = 4). d, e Comparison of the actin-bundling capacities of recombinant wt LPL, C101A LPL, and C42A LPL. Quantification of the percentages of d actin and e LPL in the pellet fraction. The calculations were performed as described above. The data are presented as the mean ± SEM (n ≥ 3; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns nonsignificant). P-values were calculated by t-test (b, c) and by two-way ANOVA (d, e). f Electron micrographs of actin bundles (>20 nm) and actin filaments (<15 nm). Recombinant wt LPL (left) or C101A LPL (right) was kept untreated (upper panels) or pretreated with 50 µM H₂O₂ (lower panels) and coincubated with G-actin for 4 h. Thereafter, the samples were prepared and imaged by TEM. Scale bar = 200 nm (n = 2)