Anti n cadherin 66219 1 ig

After culture and treatments, cells were harvested and lysed in protein extraction reagent (78501, ThermoFisher) and mixed with 1% protease inhibitor mixture at 4 °C for 30 min. Protein samples (40 μg) were separated by SDS-PAGE and transferred to PVDF membranes. Next, the membranes were blocked with 10% non-fat milk for 1 h and incubated overnight at 4 °C with primary antibodies: anti-IDH3α (15909-1-AP, Proteintech, Chicago, IL, USA), anti-HIF-1α (ab51608, Abcam, Cambridge, UK), anti-E-cadherin (60335-1-Ig, Proteintech), anti-N-cadherin (66219-1-Ig, Proteintech), anti-Vimentin (ab8978, Abcam), anti-slug (12129-1-AP, Proteintech), anti-cGAS (15102, CST, Danvers, MA, USA), anti-sting (13647, CST), anti-phospho-sting (Ser366) (19781,CST), anti-TBK1 (38066, CST), anti-phospho-TBK1 (S172) (ab109272, Abcam), anti-IRF3(ab76409, Abcam), anti-phospho-IRF3(S386)(ab76493, Abcam), anti-p65 (ab32536, Abcam), anti-phospho-p65 (S536) (ab76302, Abcam), anti-PD-L1 (ab213524, Abcam), and β-tubulin (sc-8432, Proteintech). Finally, the membranes were incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Western blotting analysis was performed using a Bio-Rad chemiluminescence imager. Original blots can be found at Supplementary Figure S5.

The specific procedures were conducted according to our previous study [22 ]. The primary immunoblotting antibodies used in this study were as follows: anti-GAPDH (10494–1-AP, Proteintech), anti-E-cadherin (20874–1-AP, Proteintech), anti-N-cadherin (66219–1-Ig), anti-PARP1 (13371–1-AP, Proteintech), anti-AKT (C67E7, Cell Signaling Technology), anti-pAKT (S473) (193H12, Cell Signaling Technology), and anti-pAKT(T308) (D25E6, Cell Signaling Technology). The antibodies of YTHDF2, LHPP, METTL3 and NKX3–1 was same with IHC. GAPDH was the internal reference.

Toluene 2,4-diisocyanate (TDI) was purchased from Junsei Chemical Co. (Tokyo, Japan). Triton X-100, sodium dodecyl sulfate (SDS), phosphatase inhibitor cocktail 2/3, protease inhibitor cocktail, 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich Chemical (St. Louis, MO, USA). SB-431542 was purchased from MedChemExpress USA (Middlesex, NJ, USA). Antibodies against anti-TGF-β1(21898-1), anti-E-cadherin (60335-1-Ig), and anti-N-cadherin (66219-1-Ig) were purchased from Proteintech (Rosemont, IL, USA). Antibodies targeting anti-p-SMAD2/3 (8828S), anti-SMAD2/3(8685S), anti-α-SMA (19245S), and anti-mouse Alexa 488 (4408S) were purchased from Cell Signaling Technology (Beverly, MA, USA). The fibronectin antibody (MA5-11981) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). Anti-vimentin (sc-6260), anti-β-actin (sc-47778), HRP-conjugated anti-rabbit IgG (sc-2357), anti-rabbit CFL 488 (sc-516248), and HRP-conjugated anti-mouse IgG (sc-516102) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, Dallas, CA, USA).