Total tak1

Small-molecule FAK inhibitor PND-1186 and TAK1 inhibitor Takinib were purchased from Selleck Chemicals (Houston, TX). LPS was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against extracellular signal-regulated kinase (ERK), phosphorylated (p)-ERK, total p38, p-p38, c-Jun N-terminal kinase (JNK), p-JNK, p-FAK, total FAK, p-TAK1, total TAK1, inhibitor of κBα (IκBα), p65 subunit of NFκB, p-p65, IκB kinase β (IKKβ), and p-IKKα/β were purchased from Cell Signaling (Danvers, MA, USA). Antibodies against GAPDH and F4/80 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Lamin B antibody was purchased from Abcam (Abcam, USA). Secondary antibodies were obtained from Santa Cruz Biotechnology.

The following antibodies used in this study were purchased from Cell Signaling: acetyl-α-tubulin (1:1000; #5335), TAB1 (1:1000; #3226), pERK (1:2000; #9101), total ERK (1:2000; #4695), pAKT-S473 (1:1000; #4060), pP38 (1:2000; #4511), pJNK (1:2000; #9255), cMYC (1:2000; #13987), pGSK3β-Ser9 (1:2000; #5558), total GSK3β (1:2000; #12456), pTAK1 (1:1000; #9339), total TAK1 (1:1000; #4505), total AKT (1:1000; #4691), pPRAS40 (1:2000; #2997), total PRAS40 (1:2000; #2610), pSmad2 (1:1000; #8828), and Myc-Tag (1:4000; #2276). Total tubulin YOL1/34 (1:2000; MCA78G) (Abcam), αHA (1:3000; #11867431001), α-Flag (1:4000; F1804) (Sigma), αTAT1 phosphoserine 237 (1:250) (Thermo Scientific), and αTAT1 (1:500; sc-167354) (Santa Cruz) were also used.

Equal amounts of protein were separated using SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were incubated with antibodies against phospho-eNOS, total-eNOS, phospho-TAK1, total-TAK1, phospho-JNK, total-JNK, phospho-p38 MAPK, total-p38 MAPK, phospho-ERK1/2, total-ERK1/2 (Cell Signaling Technology), and β-actin (Sigma-Aldrich) (Table S2). The relative density was calculated as the ratio of the intensity of the protein of interest to that of β-actin, and all band intensities were within the linear range.