P perk sc 32577

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.

Angiotensin II (Ang II) and tunicamycin (TM) were obtained from Sigma–Aldrich (St. Louis, MO). Simvastatin was purchased from Cayman Chemical Company (Ann Arbor, MI), and atorvastatin was purchased from Pfizer (New York City, NY, USA). Antibodies against β-actin(BM0627), IL-6 (BA4339-2), IL-8(BA0996), IL-1β(BA2782), MCP-1 (BA1843-2), CD68(BA3638) were from Boster Biological Technology(Pleasanton, CA). Protein kinase RNA (PKR)-like ER kinase (PERK)(sc-32577) (H-300), phosphorylated protein kinase RNA (PKR)-like ER kinase (p-PERK) (sc-32577), inositol-requiring protein-1α (IRE1α) (sc-20790), glucose-regulated protein 78 (GRP78) (sc-13968), ER marker KDEL(sc-58774), phosphorylated α-subunit of eukaryotic translation initiation factor-2 (p-EIF2α)(FL-315), and Caspase12(sc-21747) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Activating transcription factor-6α (ATF6α)(24169-1-AP) was from Proteintech Group, Inc (Chicago, USA). Antibody for CHOP(L63F7) was purchased Cell Signaling Technology (Beverly, MA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Life Technologies (Grand Island, NY). Horseradish peroxidase-conjugated secondary antibodies were from Thermo Fisher Scientific (Rockford, IL). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated.

SDS-PAGE and western blotting were performed as previously described. [28 (link)] Briefly, cell lysates were generated using RIPA buffer containing protease phosphatase inhibitor cocktail (Pierce). Cell lysates were subject to centrifugation and the protein content in supernatants was determined using the BCA Protein Assay Kit (Pierce). Proteins were resolved by 7.5%-12% SDS-PAGE, transferred to PVDF membrane and blocked with 5% bovine serum albumin. Proteins were detected with the following antibodies; cleaved Caspase 3 (MAB835) and hGRP-78 (MAB4846) from R&D Systems, BAX (ab32503) and Bcl-2 (ab7973) from Abcam, GAPDH (G8795) from Sigma Aldrich, p-PERK (sc-32577) from Santa Cruz Biotechnology, and Caspase-3 (9662), IRE-1 (3294S), CHOP (2895S), p-eIF2α (3597s), eIF2 (9722), PERK (3192S), p-P38 (9211), P38 (9212), p-Erk1/2 (9101), Erk (9102), p-SAPK-JNK (9251), and SAPK/JNK (9252) from Cell Signaling. ATF6 (MA5-16172) was purchased from Thermo Fisher Scientific. Blots were incubated with HRP-conjugated secondary antibodies followed by enhanced chemiluminescence (ECL) detection. Results were quantified by densitometry of digitized images using ImageJ software (NIH, Bethesda, MD, ver.1.43) and expressed as a ratio to a GAPDH loading control.