Ab99359

For FASN immunoprecipitation (IP) assays, 10 million empty vector or pBabe-LMP1 DG75 cells were collected for each IP and resuspended in 1 ml of radioimmunoprecipitation assay (RIPA) buffer with protease/phosphatase inhibitor cocktail (Thermo Scientific). Before addition of 10 μg of either FASN (ab99359; Abcam) or normal rabbit IgG (111-005-003; Jackson), 50 μl of cell lysate was collected and kept as the input material. Cell lysates were incubated with respective antibodies for 1 h at room temperature, with rotation, after which 30 μl of protein A magnetic beads (10001D; Invitrogen) was added. The mixture was left to incubate overnight at 4°C, with rotation. The beads were then separated with a magnetic rack and washed three times in RIPA buffer with protease/phosphatase inhibitor, each for 10 min in a 4°C ThermoMixer at 1,000 rpm. The beads were then boiled at 95°C for 8 min in 50 μl 2× Laemmli buffer, with half of the volume run on an immunoblot for FASN and half for USP2a (ab66556; Abcam) as described above. Densitometry analysis was performed on Invitrogen iBright analysis software, with signal density/area from IgG control lanes subtracted from IP lanes. IgG-normalized IP signal then was normalized to input signal density/area. Data shown are representative of three independent co-IP assays, averaged.

BMECs were collected using 0.25% trypsin and lysed in RIPA buffer (Solarbio, China) supplemented with 1% phenylmethanesulfonyl fluoride (PMSF; Pierce, USA) and 1% phosphatase inhibitor cocktail (Roche). Western blotting was performed as previously described [4 (link)], using primary antibodies against β-actin (mAbcam 8226, 1:1,000, Abcam), peroxisome proliferator-activated receptor gamma (PPARγ; EP4394(N), 1:1,000, Abcam), fatty acid synthase (FASN; ab99359, 1:2,000, Abcam), protein kinase B (Akt, #9272, 1:500, CST), pho-Akt (Ser473, 1:500, CST), mTOR (5536 T, 1:1,500, Univ), pho-mTOR (5536 T, 1:1500, Univ), ribosomal protein S6 kinase B1 (P70S6K; 2708 T, 1:1,500, Univ), pho-P70S6K (9234 T, 1:1,500, Univ), Eukaryotic Translation Initiation Factor 4E-Binding Protein 1 (4E-BP1; 9644 T, 1:1500, University), Pho-4E-BP1 (2855 T, 1:1,500, Univ), and RAI14 (EPR8518, 1:1,000, Abcam).

Protein expression was examined by western blot analysis. Briefly, cells receiving indicated treatments was washed twice with ice-cold PBS. Protein was harvested by RIPA lysis and extraction buffer (Thermo Scientific, 89900) added with protease inhibitor cocktail (Thermo Scientific, 78438). Equal amount of proteins was fractionated on 10% SDS–PAGE gel and transferred to polyvinylidence difluoride (PVD) membranes. The PVD membranes were incubated with the indicated primary and secondary antibodies. Proteins were ultimately visualized by enhanced chemiluminescence (Thermo Scientific, 32106) and autoradiography. Antibodies against β-actin, E-cadherin, Vimentin, and FASN were purchased from Abcam (ab8226, ab99359, ab8978, and ab1416).

Whole cell lysate was prepared by lysing cultured cells in radioimmunoprecipitation assay buffer (pH 8.0) containing protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland). Protein concentrations were determined using a bicinchoninic acid (BCA) kit. Proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA). The membrane was probed overnight with primary antibodies at 4° C and washed with tris-buffered saline-Tween 20 (TBST). The washed membrane was probed with secondary antibodies for 1 h at room temperature. The following antibodies were used: FASN (ab99359; 270 kDa; 1:500; Abcam, Cambridge, UK), PPAR-γ (ab59256; 54 kDa; 1:100; Abcam), Bax (ab53154; 25 kDa; 1:1000; Abcam), cleaved caspase-9 (ab2324; 40 kDa; 1:1000; Abcam), caspase-9 (ab25758; 50 kDa; 1:1000; Abcam), and Actin (ab8227; 43 kDa; 1:1000; Abcam). After several washes with TBST, the bands on the membrane were detected using the SuperSignal West Femto Maximum Sensitivity Substrate kit (Thermo Fisher Scientific, Waltham, MA, USA).