Ab184909

The chemical (2S, 3S)-1,4-Bis-sulfanylbutane-2,3-diol (DTT, CAS No. 3483-12-3) was obtained from Sigma; it is a strong reductant with the chemical formula C₄H₁₀O₂S₂ and MW of 154.25 g/mol. Its reducibility is largely due to the conformational stability after oxidation (containing disulfide bond). In this experiment, 3.09 g DTT powder was completely dissolved in 20 mL 0.01 M sodium acetate to obtain 1 M DTT stock solution, which was packed and stored at −20 °C before use.
Specific antibody against Nrf1 was made in our own laboratory [25 (link)]. All nine distinct antibodies against Nrf2 (ab62352), GCLC (ab207777), GCLM (ab126704), HO-1 (ab52947), GPX1 (ab108427), XBP1 (AB109221), ATF4 (ab184909), ATF6 (ab227830) and P4HB (ab137180) were obtained from Abcam (Cambridge, UK). The first three antibodies against TALDO (D623398), GSR (D220726) and NQO1 (D26104) were purchased from Sangon Biotech (Shanghai, China). The second three antibodies against BIP (bs-1219R), Chop (bs-20669R) and pIRE1 (bs-16698R) were from Bioss (Beijing, China). The third three antibodies against PSMB5 (A1975), PSMB6 (A4054), PSMB7 (A14771) were from ABclonal (Wuhan, China). Lastly, three antibodies to p-eIF2α (#5199) were from Cell Signaling Technology (Boston, MA, USA), p-PERK (sc-32577) from Santa CruZ (Santacruz, CA, USA), and β-actin (TA-09) from ZSGB-BIO (Beijing, China), respectively.

Brucine, ferric ammonium citrate (FAC), and glutathione (GSH) were all purchased from Sigma-Aldrich (Saint Louis, MO, USA). Ferrostatin-1 (Fer-1), liproxastin-1, and 4-phenylbutyrate (4-PBA) were all obtained from Selleck Chemicals (Houston, TX, USA). Primary antibodies against GRP78 (ab12685), ATF3 (ab254268), ATF4 (ab184909), GPX4 (ab125066), cystine-glutamate antiporter xCT (ab175186), ferritin light chain (ab69090), ferritin heavy chain (ab75972), FPN (ab78066), TF (ab82411), TFR (ab1086), ATG5 (ab108327), LC3B (ab192890), p62 (ab109012), Beclin-1 (ab207612), superoxide dismutase 1 (SOD1) (ab51254), and catalase (ab209211) were all from Abcam (Cambridge, UK). Anti-PERK (#5683) and anti-β-Actin (#4970) antibodies were from Cell Signaling Technology (Beverly, MA, USA). The other reagents were purchased from Sigma-Aldrich.

The treated human LO-2 hepatocytes were lysed to isolate proteins, which were subsequently loaded and separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the proteins were transferred onto the polyvinylidene difluoride (PVDF) membrane and incubated with 5% nonfat milk. The membrane was then incubated with primary antibody against NOX-4 (1:1000, ab133303, Abcam), NLRP3 (1:1000, ab263899, Abcam), p-eIF-2α (1:1000, ab169528, Abcam), ATF4 (1:1000, ab184909, Abcam), GADD34 (1:1000, ab236516, Abcam), CHOP (1:1000, ab11419, Abcam), NF-κB p65 (1:1000, ab288751, Abcam) or β-actin (1:1000, ab8226, Abcam), which was further incubated with an HRP-conjugated secondary antibody. Finally, the blots were developed with electrochemiluminescence (ECL) reagents and analyzed using Image J software [25 (link)].

ER-related markers, including heat shock protein family A (Hsp70) member 5 (HSPA5, also known as GRP78), DNA Damage Inducible Transcript 3 (DDIT3, also known as CHOP), activating transcription factor 4 (ATF4) and caspase12 (CASP12), were quantified by western blot. The primary antibodies were purchased from Abcam, including anti-HSPA5 (ab21685; Abcam, Cambridge, MA, USA), anti-DDIT3 (ab11419; Abcam), anti-ATF4 (ab184909; Abcam), anti-CASP12 (anti-caspase12; ab62484; Abcam), anti-PDE4A (ab200383; Abcam). Total protein was extracted using RIPA lysis buffer (Beyotime) and quantified by BCA kit (Beyotime). Protein was separated and transferred onto PVDF membranes. Protein on membranes was blocked by 5% skim milk and subsequently incubated with the primary antibodies and the secondary antibody (ab205718; Abcam). Finally, the protein bands were emerged using the ECL reagent (Beyotime) and imaged using Amersham Imager. A total of 3 independent experiments were implemented.