Fig. 2

TRX1 trapping and differential alkylation of LPL Cys-Ala mutants. a Representative immunoblots (IB) showing trapping of LPL by TRX1 C35S upon H₂O₂ treatment. PC3 cells were kept untreated or were treated with 0.1 mM H₂O₂. Thereafter, the cells were lyzed, and the trapping reaction was performed using TRX1 C35S (the trapping mutant) or TRX1 C35C (wt). TRX1-bound complexes were purified by streptavidin affinity purification and analyzed by western blotting under reducing and nonreducing conditions. Lysates were used as controls, and the other lanes show the trapping by wt TRX1 or C35S TRX1 (n = 3). b Representative IB of the trapping of different LPL Cys-Ala mutants (n = 3). HEK293 cells were transiently transfected with Cys-Ala LPL mutants, and trapping was performed as described. Lysates of LPL-expressing cells were used as controls. c Representative IB of differential alkylation of recombinant wt LPL (n = 3). Recombinant LPL was directly used or was reduced using DTT prior to differential alkylation. Thereafter, the LPL was kept untreated or was treated with 0.1 mM H₂O₂ for 15 min. Following this step, the samples were subjected to differential alkylation. Then, the samples were run on 6% nonreducing or reducing SDS-polyacrylamide gels and immunoblotted for LPL (n = 4). d Representative IB of differential alkylation of recombinant wt, C42A and C101A LPL after pre-reduction with DTT (n = 3). The samples were prepared and immunoblotted for LPL. The red arrow indicates the upper band for the oxidized form of LPL, while the black arrows indicate the lower band for the reduced form of LPL. e TRX1 trapping in PC3 cells and f PBTs. The cells were treated with the indicated concentrations of H₂O₂ and subjected to TRX1 kinetic trapping. Representative IBs stained for LPL and PRX1 are shown (n = 3)