Control mouse igg

The following primary antibodies were employed in study: rabbit anti-GAPDH [1:400; Cell Signaling Technology (CST); Catalog number-5174S)], rabbit anti-LC3B (1:400; CST; 2775S), rabbit anti-beclin1 (1:350; CST; 5174S), rabbit anti-ATG7 (1:400; CST; 8558S), rabbit anti-P62 (1:400; CST; 5114S), anti-cleaved caspase 8 (1:200; CST; 9748), anti-phospho BRAF (1:1000; CST; 2696T), anti-phospho-MEK (1:400; CST; 9154T), Mouse anti-beta actin (1:10,000; Abcam; ab184220), rabbit anti-LAMP-1(1:250; Abcam; ab25630), rat anti-LAMP-2 (1:250; Abcam; ab25631), rabbit anti-Fas L (1:100; Abcam; ab15285), rabbit anti-caspase 3 (1:400; Thermo Fisher; 4331182), rabbit anti-Fas (1:100; Bio legend; 305611), and mouse anti-a2V (Covance, Denver, USA). For isotype-control antibodies, control mouse IgG (R&D Systems) and rabbit IgG isotype (Invitrogen) were used. Secondary antibodies were as follows: goat anti-rabbit IgG-FITC, donkey anti-mouse IgG AF-594, donkey anti-rabbit IgG AF-594 (Invitrogen), rabbit anti-rat IgG-FITC (Abcam), donkey anti-rabbit IRDye-800CW, and donkey anti-mouse IRDye-680 CW (LI-COR Bioscience, Lincoln, NE).

An in vitro invasion assay was performed in triplicate using Growth Factor Reduced BD BioCoat Matrigel Invasion Chambers (BD Biosciences) according to the manufacturer’s instructions. Briefly, 3 × 10⁴ cells were seeded in the upper chamber with conditioned medium from PrSC treated with or without 100ng ml^-1 rFABP4, and the presence or absence of 10  μg ml^-1 of IL-8 blocking antibody, IL-6 blocking antibody, control goat IgG, or control mouse IgG (R&D Systems, Minneapolis, MN, USA). In the siRNA experiments, cells were treated with 50 nM FABP4 siRNAs for 24 hours before being seeded in the chambers. Subsequently, 20% FBS DMEM was placed in the lower chamber, followed by incubation for 24 hours. In some experiments, 1 × 10⁴ PrSC were seeded in the lower chamber with optimal medium. Sera from mice were collected, clarified by filtration (SLGV004SL; Millipore, Billerica, MA, USA), and used for ex vivo cell invasion assays. Briefly, 3 × 10⁴ cells were seeded in the upper chamber with medium containing 5% FBS or 5% mouse serum with or without 10  μg ml^-1 of IL-8 blocking antibody or 30 μM of BMS309403. DMEM with 20% FBS was placed in the lower chamber. Then, the non-invading cells in the upper chamber were removed and the membranes were stained with a Diff-Quik cell-staining kit (Sysmex, Kobe, Japan) to count the invading cells. The experiments were performed twice each in triplicate.

Primary antibodies were as follows: mouse anti-a2V (Covance, Denver, PA); rabbit anti-GAPDH (Cell signaling, Danvers, MA); rat anti-F4/80, rabbit anti-LC3B, rabbit anti-LAMP-1, rat anti-LAMP-2, rabbit anti-ATG3, rabbit anti-ATG4, rabbit anti-ATG7, rabbit anti-NF-κB p65 (all from Abcam). Secondary antibodies were as follows: goat anti-rabbit IgG-FITC, -mouse IgG AF-594, -rabbit IgG AF-594 (Invitrogen), rabbit anti-rat IgG-FITC (Abcam), goat anti-rabbit, -rat, -mouse IgG-HRP (Santa Cruz Biotechnology), donkey anti-rabbit IRDye-800CW (LI-COR Bioscience, Lincoln, NE) and EnVision + dual link System-Horseradish peroxidase (HRP) (Dako). Isotype control antibodies were as follows: control mouse IgG (R&D Systems); rat IgG isotype, mouse IgG isotype and rabbit IgG isotype (Abcam).