Anti fyn

For co-immunoprecipitation assay, collected cells were lysed in the lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% NP-40, 1 mM sodium fluoride and 1 mM sodium orthovanadate. Insoluble proteins (500 μg per sample) were incubated with anti-Fyn antibody (Santa Cruz, sc-16) and Protein A/G Plus Agarose beads (Santa Cruz) for 14 h at 4°C with constant rotation. Immunocomplexes were washed four times with 0.1% Triton-100 in PBS and eluted by boiling in the Laemmli buffer. Primary antibodies used for immunoblot were listed as follows: anti-PTPRZ1 (BD Biosciences, 610179, 1: 1,000); anti-SOX2 (Bethyl Laboratories, A301-739A, 1: 1,000); anti-Phospho-AKT (p-Ser473, Cell Signaling, #4060, 1: 2,000); anti-AKT (Cell Signaling, #2966, 1: 500); anti-CD133 (Millipore, MAB4399, 1: 500); anti-GFAP (Covance, PRB-571C, 1: 1,000); anti-MAP2 (Covance, SMI-52R, 1: 1,000); anti-p-SFK (Tyr416, Cell Signaling, #6943, 1: 500); anti-Fyn (Cell Signaling, #9320, 1: 500); anti-Tubulin (Sigma, T9026, 1: 10,000); and anti-GAPDH (Cell Signaling, #2118, 1: 2,000). Representative uncropped immunoblots were presented in Supplementary Fig. 12. All immunoblot results were independently repeated for at least three times.

Erlotinib was kindly provided by F. Hoffman-La Roche Ltd, gefitinib was provided by AstraZeneca Inc; VS-4718 was provided by Verastem Inc; afatinib, osimertinib, lapatinib, AZD4547, and BIBF1120 were purchased from Selleck Chemicals; SU11274 and picropodophyllin were from Carbiochem; dasatinib was from Bio Vision; SB203580 was from Cayman Chemical; sorafenib was acquired from Toronto Research Chemicals Inc, cisplatin was from Bristol-Myers Squibb Company; and PD173074 was from Sigma-Aldrich.
Anti-HER2 and anti-pHER2 antibodies were purchased from Merck Millipore Corporation, anti-EGFR, anti-pEGFR, anti-pHER3, anti-HER4, anti-pHER4, anti-pc-Met, anti-IGF1Rβ, anti-pIGF1Rβ, anti-PDGFRβ, anti-pPDGFRβ, anti-FGFR1, anti-pFGFR, anti-ERK1/2, anti-pERK1/2, anti-AKT, anti-pAKT, anti-STAT3, anti-pSTAT3, anti-PTEN, anti-SRC, anti-FYN, anti-LYN, anti-YES, anti-LCK, anti-pSRC family (Y416), anti-pSRC (Y527), anti-FAK, anti-pFAK (Y397), anti-pFAK (Y576/577), anti-pFAK (Y925) and anti- EGFR (del E746-A750) antibody were from Cell Signaling Technology, anti-HER3 and anti-c-Met were from Santa Cruz Biotechnology Inc, anti-β-actin was from Abcam, Inc., and anti-α-tubulin was from Sigma-Aldrich.

Proteins were extracted from cells via radioimmunoprecipitation assay buffer (Beyotime Biotechnology) after different treatments, then equally separated by 10% SDS-PAGE gel, and ultimately transferred into nitrocellulose membranes (GE Healthcare Life Sciences, Beijing, China). Membranes were blocked in 5% nonfat milk for 1 h, and incubated overnight at 4°C with anti-phosphorylated-AKT (P-AKT, 1:1,000, Cell Signaling Technology), anti-AKT (1:1,000, Cell Signaling Technology), anti-P-GSK3β (1:1,000, Cell Signaling Technology), anti-GSK3β (1:1,000, Cell Signaling Technology), anti-FYN (1:1,000), anti-NRF2 (1:1,000), anti-BAX (1:1,000, Cell Signaling Technology), anti-BCL-2 (1:1,000, Abcam), anti-CLEAVED CASPASE-3 (C-CASPASE 3, 1:1,000, Cell Signaling Technology), anti-GAPDH (1:1,000, Servicebio technology), and anti-β-ACTIN (1:2000, Servicebio technology). The catalogs of antibodies are shown in Supplementary Table S1.

Hyp with a purity of ≥ 98% was purchased from Nanjing Zelang Medical Technological Co., Ltd (Nanjing, China), while CCl4 and tert-butylhydroquinone (t-BHQ) were obtained from Sigma Chemical (St. Louis, MO, USA). Moreover, rabbit antibodies anti-PHLPP2 (Cat. #SAB1300919; Sigma), anti-AKT (Cat. #9272; Cell Signaling Technology, Danvers, MA, USA), anti-phosphorylated (p)-AKT (S473) (Cat. #4060; Cell Signaling Technology), anti-p-AKT (Thr308) (Cat. #2965; Cell Signaling Technology), anti-GSK-3β (clone #3D10; Cat. #12456; Cell Signaling Technology), anti-p-GSK-3β (Ser9) (Cat. #9322; Cell Signaling Technology), anti-Fyn (Cat. #4023; Cell Signaling Technology), anti-Histone H2 (Cat. #2595; Cell Signaling Technology), anti-Nrf2 (pS40) (Cat. #ab89443; Abcam, Cambridge, MA, USA), and anti-Nrf2 (Cat. #ab89443, Abcam), and anti-HO-1 (Cat. #SC-10789; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p-Fyn (Thr) (Cat. #sc-5267; Santa Cruz Biotechnology), and a mouse antibody anti-GAPDH (Cat. #SC-365062; Santa Cruz Biotechnology) were purchased from the named companies, respectively.