Lysis buffer (200 μl/well) was used to lyse HepG2 cells. Lysis buffer was composed of
Triton X-100 (1%), Tris (50 mM, pH=7.6) and NaCl (150 mM), with inhibitors of phosphatases
and proteases. sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 7.5%)
was used to separate 40 μg of the total extracted protein. Then Western blotting was done
as demonstrated by Moeschel et al. (10 (link)). Following the application of antibodies for
western blotting anti-PRDX1 (ab109498), anti-beta actin (ab115777), anti
caspase-3 (ab13847 and ab32042), anti-cleave caspase-9 (ab202068 and ab25758), anti-PARP-1
(ab191217), anti-Bim (ab7888), antiFis1 (ab189846), anti-APaf-1 (ab254248), anticytochrome
c (ab133504), anti-Bcl-2 (ab182858), anti-Bax (ab3191), anti-DRP1 (ab184247) and antiDyn2
(ab65556; all purchased from AbCam, UK). Nitrocellulose was blocked using skimmed milk
(5%) or BSA (2%, both from Merck, Germany) for two hours. Subsequently, membranes were
incubated with primary antibodies at 4°C overnight. Before incubation with secondary
antibody, washing was performed (four times for 10 minutes), followed by appropriate
conjugated secondary incubation for one hour. For visualizing expression level of
proteins, enhanced chemiluminescence was performed.
Triton X-100 (1%), Tris (50 mM, pH=7.6) and NaCl (150 mM), with inhibitors of phosphatases
and proteases. sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 7.5%)
was used to separate 40 μg of the total extracted protein. Then Western blotting was done
as demonstrated by Moeschel et al. (10 (link)). Following the application of antibodies for
western blotting anti-PRDX1 (ab109498), anti-beta actin (ab115777), anti
caspase-3 (ab13847 and ab32042), anti-cleave caspase-9 (ab202068 and ab25758), anti-PARP-1
(ab191217), anti-Bim (ab7888), antiFis1 (ab189846), anti-APaf-1 (ab254248), anticytochrome
c (ab133504), anti-Bcl-2 (ab182858), anti-Bax (ab3191), anti-DRP1 (ab184247) and antiDyn2
(ab65556; all purchased from AbCam, UK). Nitrocellulose was blocked using skimmed milk
(5%) or BSA (2%, both from Merck, Germany) for two hours. Subsequently, membranes were
incubated with primary antibodies at 4°C overnight. Before incubation with secondary
antibody, washing was performed (four times for 10 minutes), followed by appropriate
conjugated secondary incubation for one hour. For visualizing expression level of
proteins, enhanced chemiluminescence was performed.