Beta3 tubulin

Erastin (cat. no. E7781), ferrostatin‐1 (Fer‐1; cat. SML0583), and liproxstatin‐1 (Lpx‐1; cat. SML1414) were purchased from Sigma‐Aldrich (Saint Louis, MO, USA). (1S,3R)‐RSL3 (cat. HY‐100218A) was purchased from MedChem Express (Monmouth Junction, NJ, USA). EGTA‐AM (cat. 20401) and BAPTA‐AM (cat. 15551) were from Cayman Chemical (Ann Arbor, MI, USA). Antibodies against MNX1 (Hb9) were from Developmental Studies Hybridoma Band (cat. 81.5C10) (Iowa City, IA, USA), beta3‐tubulin was from Cell Signaling Technology (cat. 5568) (Danvers, MA, USA), and actin was from Santa Cruz Biotechnology (cat. no. sc‐1616‐R) (Dallas, TX, USA). Antibodies against choline acetyltransferase (ChAT) and GPx4 were from Abcam (cat. ab178859 and ab125066, respectively) (Cambridge, MA, USA). Secondary antibodies against mouse IgG and rabbit IgG were from LI‐COR Biotechnology (cat. 925‐68070 and 925‐68071) (Lincoln, NE, USA).

Cells were treated as previously described. Then, they were lysed according to Pacifici et al. [46 (link)]. Briefly, cells were lysed in ice-cold buffer composed by: 20 mM Tris (pH 7.6), 137 mM NaCl, 1.5% NP40, 1 mM MgCl₂, 1 mM CaCl₂, Glycerol 10%, 2 mM phenylmethylsulfonyl fluoride (PMSF), phosphatase inhibitor cocktails (Sigma Aldrich, Saint Louis, MO, USA), and protease inhibitor cocktail tablets (Roche Diagnostics GmbH, Mannheim, Germany), and maintained in ice for 30 min. Then, they were centrifuged at 14,000× g rpm for 25 min. The collected supernatants were loaded on a 4–12% pre-cast gel (Thermo Scientific, Waltham, MA, USA) and transferred on a nitrocellulose membrane by using the Trans Turbo Blot system (Bio-Rad Laboratories, MI, Italy). Membranes were probed with the following primary antibodies: caspase-3, PARP-1, Beta-3 tubulin, Tyrosine hydroxylase, pERK1/2, total ERK1/2 and actin (1:1000 Cell Signaling Technologies, Denvers, MA, USA), vinculin (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA).
The antigen antibody complexes were detected with enhanced chemiluminescence (ECL) reagent (GE Healthcare, Little Chalfont, UK) and quantified by using the Gel DocTM XR + with Image LabTM Software (Bio-Rad Laboratories, MI, Italy).