Ab33899

Total protein extracts were prepared according to standard methods. Forty micrograms of protein extract was fractioned by SDS-PAGE and transferred onto Hybond nitrocellulose filters (RPN 303D, GE Healthcare, Milan, Italy). Filters were blocked in PBS-Tween 20 and 5% skim milk and incubated overnight with the following primary antibodies: mouse monoclonal anti-BCL-2 (sc-509, Santa Cruz Biotechnology, Dallas, TX, USA), anti-KIT (#3308; Cell Signaling Technology, Danvers, MT, USA), and anti-MYC (ab32; Abcam, Cambridge, United Kingdom); rabbit polyclonal anti-AKT(S473) (sc-9271, Santa Cruz Biotechnology), anti-BRAF (ab33899, Abcam,), anti-ERK1/2 (T202/Y204) (#9101, Cell Signaling Technology), anti-γ-H2AX (ab11174, Abcam), anti-p21^waf1 (ab7960, Abcam), and anti PARP-1 (#9542, Cell Signaling Technology). Mouse monoclonal anti-Vinculin (VCL, V9131, Sigma-Aldrich, Milan, Italy) or anti-β-Actin (Ab8226, Abcam) antibodies were used to ensure equal protein loading. The filters were then probed with secondary peroxidase-linked whole antibodies (GE Healthcare, Milan, Italy) and subjected to autoradiography using the Novex^® Enhanced Chemoluminescent Horseradish Peroxidase detection system (Thermo Fisher Scientific, Monza, Italy). Films were scanned (ImageScanner III, GE Healthcare, Milan, Italy) and images were processed by Photoshop7.0.1 or analyzed using ImageJ 1.46r.

Cell lysates were heat denatured, separated by SDS‒PAGE, transferred to PVDF membranes (abs931, Absin, Shanghai, China), and blocked with 5% skimmed milk. Then, membranes were incubated, respectively with anti-BRAF (1:1000, ab33899), anti-GPX4 (1:1000, ab125066), anti-PUMA (1:1000, ab9643), anti-LC3B (1:2000, ab192890), anti-MLKL (1:500, ab184718), anti-phospho-MLKL (1:1000, ab196436), anti-ERK1/2 (1:1000, ab184699), anti-Ubiquitin (1:1000, ab134953), anti-AMPKα (1:2000, ab32047), anti-p62 (1:1000, ab109012), anti-Beclin1 (1:2000, ab207612) primary antibodies from Abcam (UK), anti-Alpha Tubulin (1:1000, 11224-1-AP), anti-FOXO3a (1:1000, 10849-1-AP), anti- SLC7A11 (1:1000, 26864-1-AP), ati-H3 (1:1000, 17168-1-AP) primary antibodies from Proteintech (IL, USA), anti-Cleaved Caspase-3 (1:1000, #9661), anti-phospho-Beclin1 (1:1000, #14717), anti-phosphor-AMPKα (1:1000, #2535), anti-phospho-FOXO3a (1:1000, #64616), anti-phospho-ERK1/2 (1:1000, #4370) primary antibodies from Cell Signaling Technology (MA, USA) and anti-ATG5 (1:1000, ET1611-38) from HUABIO (Hangzhou, China) overnight at 4 °C and anti-rabbit (1:5000, ab205718, absin, Shanghai, China) or anti-mouse (1:5000, abs20039, absin, Shanghai, China) secondary antibodies for 1 h at room temperature. Immunoblots were visualized using an ECL detection reagent (BMU101-CN, Abbkine Scientific, Wuhan, China).

Spinal cord tissue, or neurons were fixed in 4% PFA for 20 min. Thenwe were incubated with primary rabbit anti‐SRF antibody (1:200 diluted by PBS, CST, D71A9, USA), anti‐Ras antibody (1:500 diluted by PBS, Abcam, ab52939, USA), anti‐Raf antibody (1:250 diluted by PBS, Abcam, ab33899, USA), anti‐cofilin antibody (1:300 diluted by PBS, Abcam, ab42824, USA), anti‐GAP43 antibody (1:500 diluted by PBS, Abcam, ab75810, USA) and anti‐NF antibody (1:50 diluted by PBS, ZSGB‐BIO, 18703E05, China) overnight at 4°C, incubator with DAPI (1:700 diluted by PBS, Solarbio, China) 10 min, and washed with PBS for 5 times (3 min each time). After washing, the secondary antibody (1:500, Abcam, USA) was applied, and the mixture was incubated at 37°C for 1 h. Images were recorded using a microscope.