Untreated or specifically treated cells were washed twice with cold PBS and harvested in RIPA lysis buffer supplemented with 1% protease inhibitor. Cells were scrapped, lysates were collected, homogenized with syringe and needle, and centrifuged at 30,000 x g for 5 min at 4 °C. Clear lysate was transferred to a new tube. Protein concentration of lysate was determined using the DC assay (BioRad). Protein samples were resolved by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare).
Primary antibodies used: CBS (1:1000, sc-133154, Santa Cruz Biotechnology), MPST (1:4000, HPA001240, Sigma Aldrich), CTH (MEF cells, 1:4000), CTH (HeLa cells, 1:1000, sc-374249, Santa Cruz Biotechnology), p-ERK (1:1000, sc-7383, Santa Cruz Biotechnology), total-ERK (1:1000, sc-271269, Santa Cruz Biotechnology), DJ-1 (1:250, sc-55572, Santa Cruz Biotechnology), DJ-1 Oxidized At C106 (1:1000, HCA024, BioRad) and β-tubulin (1:5000, T0198, Sigma Aldrich). Species-specific horseradish-conjugated secondary antibodies (1:5000, Santa Cruz Biotechnology) were used for antigen detection and visualized using Clarity™ Western ECL Substrate (BioRad) on a G:Box Chemi-XT4 (Syngene).
Primary antibodies used: CBS (1:1000, sc-133154, Santa Cruz Biotechnology), MPST (1:4000, HPA001240, Sigma Aldrich), CTH (MEF cells, 1:4000), CTH (HeLa cells, 1:1000, sc-374249, Santa Cruz Biotechnology), p-ERK (1:1000, sc-7383, Santa Cruz Biotechnology), total-ERK (1:1000, sc-271269, Santa Cruz Biotechnology), DJ-1 (1:250, sc-55572, Santa Cruz Biotechnology), DJ-1 Oxidized At C106 (1:1000, HCA024, BioRad) and β-tubulin (1:5000, T0198, Sigma Aldrich). Species-specific horseradish-conjugated secondary antibodies (1:5000, Santa Cruz Biotechnology) were used for antigen detection and visualized using Clarity™ Western ECL Substrate (BioRad) on a G:Box Chemi-XT4 (Syngene).