Anti fak

Protein extracts from NSCLC cell lines and fresh frozen lung tissue samples were prepared in lysis buffer (0.5% Triton X-100, 50 mM β-glycerophosphate, pH 7.2, 0.1 mM dithiothreitol, 2 µg/mL leupeptin, and 2 µg/mL aprotinin). Extracts were resolved on 10% SDS-PAGE gels and transferred onto nitrocellulose membranes. Membranes were blocked in Tris-buffered saline (TTBS) containing 10 mM Tris-Cl (pH 7.4), 140 mM NaCl, 0.1% Tween 20, and 3% nonfat dry milk for 1 hour and incubated with TTBS containing indicated antibodies at 0.5 µg/mL for 12–16 hours at 4 ^oC. The following antibodies were used for immunoblotting: anti-KCNF1 (Sigma, HPA014738), anti-ITGB4 (Abcam, ab182120), anti-HA (Cell Signaling, 2367), anti-His (Proteintech, 10001-0-AP), anti-phospho-FAK (Tyr397, Invitrogen, 44624 G), anti-FAK (Invitrogen, AHO0502), anti-phospho-AKT (pS473, Cell Signaling, 9271), anti-AKT (Cell Signaling, 9272), anti-caspase 3, -caspase 7, and -caspase 9 (Cell Signaling: 9662, 9492, 9508), anti-GAPDH (Cell Signaling, 5174), and anti-Actin (Sigma, A2066). The membranes were extensively washed in TTBS and bound antibodies were visualized with horseradish peroxidase (HRP)-coupled secondary antibodies and ECL western blotting detection reagent (Amersham, RPN2106).

Anti‐DGKα (Cat# H00001606‐B01P), anti‐CD204 (Cat# PAB30252), and anti‐Gli1 (Cat# MAB16740) antibodies were provided by Abnova. Anti‐FAK (Cat# 39−6500) antibody was obtained from Invitrogen. Antibodies against CCL22 (Cat# ab217351) and CCR4 (Cat# ab83250) were obtained from Abcam. Antibodies against AKT (Cat# 9272) and SuFu (Cat# 2522) were obtained from CST. Antibody against Lamin A/C (Cat# A17319) was obtained from ABclonal. pGli1 Ser¹¹²/Thr¹¹⁵/Ser¹¹⁶ antibody was synthesized from ABclonal.

Western blot analysis was performed as described previously [21 (link)]. The primary antibodies used for Western blot analyses included anti-phospho-Src (Tyr418) (Invitrogen, Carlsbad, CA, USA), anti-phospho-FAK (Tyr576) (Invitrogen), anti-FAK (Invitrogen), anti-GAPDH (Invitrogen), anti-phospho-EGFR (Tyr1068) (Cell Signaling, Beverly, MA, USA), anti-phospho-STAT3 (Tyr705) (Cell Signaling), anti-phospho-PI3K (Tyr458) (Cell Signaling), anti-AKT (Cell Signaling), anti-phospho-SAPK/JNK (Thr183/Tyr185) (Cell Signaling), anti-SAPK/JNK (Cell Signaling), anti-phospho-Paxillin (Tyr118) (Cell Signaling), anti phosphor-p130Cas (Tyr410) (Cell Signaling), anti-EGFR (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT3 (Santa Cruz Biotechnology), anti-PI3K (Santa Cruz Biotechnology), anti-phospho-MEK1/2 (Ser218/Ser222) (Santa Cruz Biotechnology), anti-MEK (Santa Cruz Biotechnology), anti-phospho-ERK (Tyr204) (Santa Cruz Biotechnology), anti-ERK2 (Santa Cruz Biotechnology), anti-Paxillin (Santa Cruz Biotechnology), anti-p130 Cas (Santa Cruz Biotechnology), anti-phospho-AKT (Ser473) (Millipore, Billerica, MA, USA)