β actin c 4 sc 47778

GN (≥98% purity) was obtained from ChemFaces (Wuhan, China). Antibodies against SREBP-1c (H-160; sc-8984), PPARα (H-2; sc-398394), PGC-1α (H-300; sc-13067), IκBα (H-4; sc-164), NF-κB p65 (F-6; sc-8008p65), SIRT1 (H-300; sc-15404), AMPK α1/2 (D-6; sc-74461), and β-actin (C4; sc-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Specific primary antibodies against p-ACC (Ser79; #3661s) and ACC (C83B10; #3676s) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against CYP2E1 (ab28146) and p-AMPK α1 (Thr172; PA5-17831) were purchased from Abcam (Cambridge, MA, USA) and Invitrogen (Rockford, IL, USA), respectively. Ethanol and Ex52735 were obtained from Sigma (St. Louis, MO, USA).

For cultured cells, cells were washed twice with PBS and lysed with cold RIPA lysis buffer containing protease inhibitors (PMSF [phenylmethylsulphonyl fluoride] 1 mmol/L and leupeptin 0.1 g/L). Cell lysates were collected from culture plates using a rubber policeman, and protein was collected by centrifugation. Protein concentrations were determined by BCA (bicinchoninic acid) protein assay (Pierce, Rockford, IL, USA). Aliquots of 40 μg of proteins were boiled in 2× loading buffer (0.1 M Tris-Cl, pH 6.8, 4% SDS, 0.2% bromophenyl blue, and 20% glycerol) for 10 minutes, loaded into 10% Tris–HCl polyacrylamide gels, and transferred electrophoretically to Immobilon-P membrane (Millipore Corporation, Billerica, MA, USA). Membranes were incubated with primary antibodies and appropriate HRP secondary antibodies. Membranes were additionally probed with an antibody against β-actin to ensure equal loading of protein between samples. Detection was performed with chemiluminescent agents (Pierce). Derlin-1 (N-20, sc-46913), Beclin1 (H-300, sc-11427), GRP78 (H-129, sc-13968) and β-actin (C4, sc-47778) antibodies were from Santa Cruz Biotechnology, p62 (D5E2, 8025S), and LC3A/B (4108 s) antibodies were from Cell Signaling Technology.

Caerulein (CAE) was purchased from Sigma-Aldrich (St. Louis, MO, United States). Rabbit monoclonal antibodies against OPA1 (ab157457, Abcam Plc, Cambridge, United Kingdom), DRP1 (D6C7 rabbit mAb; Cell Signaling), LC3B [LC3B antibody (2775)]; Cell Signaling Technology, MA, United States), p62 [SQSTM1 (P-15): sc-10117; Santa Cruz Biotechnology, Santa Cruz, CA, United States)], VMP1 (D1y3E, Cell Signaling Technology, MA, United States); mouse monoclonal antibodies against Parkin (P6248, Sigma-Aldrich, St. Louis, MO, United States), V5 [13202 V5-Tag (D3H8Q) rabbit mAB-Cell Signaling)], β-actin [β-actin (C4): sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA, United States)], beta tubulin (ab131205, Abcam Plc, Cambridge, United Kingdom); rabbit anti-goat antibody (Santa Cruz Biotechnology, Santa Cruz, CA, United States); goat policlonal antibodies against VDAC-1 (D-16 sc-32063; Santa Cruz Biotechnology, Santa Cruz, CA, United States); rabbit anti-mouse antibody [(315-035-048) Jackson InmunoResearch, Baltimore Pike, United States]; goat anti-rabbit antibody [(GAR):170-5046; Bio-Rad, CA, United States]. Other reagents, enzymes, and chemicals were of reagent grade and also from Sigma-Aldrich.

HEL cells and leucocytes from MPN patients were used for immunoblotting assays. Total proteins were isolated with radioimmunoprecipitation assay (RIPA) buffer and quantified as previously described.²⁸ 70 µg of each cell lysate was loaded, resolved through SDS‐PAGE 6%‐12% gel and electroblotted onto 0.2 µm PVDF membranes (GE Healthcare, Chicago, IL, USA). After blocking with 5% non‐fat milk (Sigma‐Aldrich, Thermo Fisher Scientific, Waltham, MA, USA), membranes were incubated 2 hrs at RT with Bcl‐xL (H‐5 sc‐8392) or overnight at 4°C with Phospho‐Jak2 (Tyr) (3771, Cell Signaling Technology, Danvers, MA, USA), JAK2 (C‐14 sc‐34479), GAPDH (A‐3 sc‐137179) and β‐Actin (C4 sc‐47778) [Santa Cruz Biotechnology, Dallas, TX, USA] primary antibodies with a dilution of 1:2000. Then, membranes were incubated with specific horseradish peroxidase (HRP)‐conjugated secondary antibodies: anti‐rabbit (sc‐2357), antimouse (sc‐2005) and anti‐goat (sc‐2354) [Santa Cruz Biotechnology, Dallas, TX, USA] in a dilution of 1:7000 for 1 hour at RT. Immunoreactive bands were visualized by Clarity Western ECL Substrate (Bio‐Rad, Hercules, CA, USA) and acquired by the Image Lab program (Bio‐Rad, Hercules, CA, USA).

Cryptotanshinone (≥98% purity) was obtained from ChemFaces (Wuhan, China). Antibodies against SREBP-1c (H-160; sc-8984), PPARα (H-2; sc-398394), IkBα (H-4; sc-164), NFκB p65 (F-6; sc-8008p65), AMPK α1/2 (D-6; sc-74461), SIRT1 (H-300; sc-15404), and β-actin (C4; sc-47778) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Specific primary antibodies against p-ACC (Ser79; #3661s) and ACC (C83B10; #3676s) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against CYP2E1 (ab28146) and pAMPK α1 (Thr172; PA5-17831) were purchased from Abcam (Cambridge, MA, USA) and Invitrogen (Rockford, IL, USA), respectively. The Nrf2 antibody (NBP1-32822) was obtained from Novus Biologicals (Centennial, CO, USA). Ethanol, Ex52735, and compound C were obtained from Sigma (St. Louis, MO, USA).

The antibodies used for immunoblotting include: mouse monoclonal antibodies (mAbs) c-Myc antibody (sc-56634) and β-actin (C4) (sc-47778) (both purchased from Santa Cruz Biotechnology, Dallas, TX, USA); rabbit polyclonal antibodies against NP (GTX125989) was purchased from GeneTex (Taiwan); anti-TBK1 (ab40676), and anti-TBK1 (phosphor S172) (ab109272) antibodies were obtained from Abcam (Cambridge, MA, USA). Rabbit polyclonal antibody against acetylated-lysine (9441), mouse mAb against HDAC1 (10E2) (5356), p-IRF3 (4947), IRF3 (4302), p-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (4370), p44/42 MAPK (Erk1/2) (9102), and rabbit mAb HA-Tag (c29F4) (28) were from Cell Signaling Technology (Beverly, MA, USA).