Anti-IRTKS antibody (ab226344, 1:5000) for immunoblotting and anti-IRTKS antibody (ab211301, 1:600) for IHC were obtained from Abcam (Cambridge, MA, USA). Anti-AKT antibody (#4691, 1:1000) and anti-pAKT (Ser473) antibody (#4060, 1:2000) were from Cell Signaling Technology (Beverly, MA, USA). Anti-FGFR1 antibody (60325, 1:2000) was from Proteintech (Rosemont, IL, USA). Anti-β-actin antibody (A1978, 1:10000) was from Sigma (St. Louis, MO, USA). Secondary antibodies (#7074/7076, 1:10000) were purchased from Cell Signaling Technology.
IRTKS-3*FLAG, encoding full-length IRTKS with three N-terminal FLAGs, was synthesized and cloned into the vector pLVX-Puro-CMV by Vigene Biosciences (Jinan, Shandong, China). Two small interfering RNAs (siRNAs) against IRTKS were used: siRNA-1 (sense, 5′-GCA CCU ACC GGA AUG UUA UTT-3′ and anti-sense, 5′-AUA ACA UUC CGG UAG GUG CTT-3′) and siRNA-2 (sense, 5′-CCA GUC CCU UGA UCG AUA UTT-3′ and anti-sense, 5′-AUA UCG AUC AAG GGA CUG GTT-3′). In addition, siRNA-NC (sense, 5′-UUC UCC GAA CGU GUC ACG UTT-3′ and anti-sense, 5′-ACG UGA CAC GUU CGG AGA ATT-3′) was used as a control. All siRNAs were designed and chemically synthesized by GenePharma (Suzhou, Jiangsu, China).
IRTKS-3*FLAG, encoding full-length IRTKS with three N-terminal FLAGs, was synthesized and cloned into the vector pLVX-Puro-CMV by Vigene Biosciences (Jinan, Shandong, China). Two small interfering RNAs (siRNAs) against IRTKS were used: siRNA-1 (sense, 5′-GCA CCU ACC GGA AUG UUA UTT-3′ and anti-sense, 5′-AUA ACA UUC CGG UAG GUG CTT-3′) and siRNA-2 (sense, 5′-CCA GUC CCU UGA UCG AUA UTT-3′ and anti-sense, 5′-AUA UCG AUC AAG GGA CUG GTT-3′). In addition, siRNA-NC (sense, 5′-UUC UCC GAA CGU GUC ACG UTT-3′ and anti-sense, 5′-ACG UGA CAC GUU CGG AGA ATT-3′) was used as a control. All siRNAs were designed and chemically synthesized by GenePharma (Suzhou, Jiangsu, China).