Ab140915

Deparaffinization and protein extraction of FFPE tissue was carried out using Qproteome FFPE Tissue Kit (Qiagen, Hilden, Germany; #1042481) following manufacturer’s instructions. Extracted proteins were determined by Bradford assay (BIO-RAD, CA, USA; #5000205). Quantified proteins were mixed with 5 × SDS-PAGE loading buffer (Biosesang, Seongnam, Korea; S2002) in concentration of 2 μg/μL and boiled at 95 °C for 5 min. Equal quantities of protein were separated to SDS-PAGE gel and transferred to nitrocellulose membranes (BIO-RAD; #1704158). Membranes were blocked by incubation in 5% skim milk in Tris-buffered saline (TBS) with 0.1% Tween-20 and probed with antibody against ATX (1:1000; Abcam, Cambridge, UK; ab140915), LPA1(1:2000; Abcam; ab166903), and LPA2(1:1000; Abcam; ab38322) diluted in 1% BSA in TBS, 0.1% Tween 20, and 0.02% NaN₃. The membranes were washed and then incubated with secondary antibodies (HRP conjugated anti-mouse IgG, or anti-rabbit IgG) (1:20,000; Santa Cruz, TX, USA) for 1 h at room temperature. The bands were visualized using WesternBright ECL (Advansata, CA, USA; K-12045-D50) after washing the membrane and exposed to X-ray film.

The paraffin-embedded sections were deparaffinized and rehydrated, which was followed by antigen retrieval in boiling citrate buffer for 30 min. The sections were surrounded with a hydrophobic pen and rinsed with PBS. The sections were then permeabilized with 0.1% Triton X100 for 5 min in goat serum. The sections were blocked with 10% goat serum for 1 h and then incubated overnight at 4 °C with mouse anti-ATX (ab140915, Abcam, Waltham, MA, USA), mouse anti-lysophosphatidic acid (#504B3, Echelon Biosciences Inc., Logan, UT, USA), anti-phospho-ERK (#9101, Cell Signaling Technology, Inc. Waltham, MA, USA), PCNA (#13-3900, Invitrogen, Waltham, MA, USA), and anti-smooth muscle actin (#ab5694, Abcam, Waltham, MA, USA) as primary antibodies. The sections were then incubated with AlexaFluor 488 goat anti-mouse (#A28175, ThermoFisher Scientific, Waltham, MA, USA) and AlexaFlour 594 goat anti-rabbit (#A11012, ThermoFisher Scientific, Waltham, MA, USA) before then being rinsed with PBS. After 1 h, the sections were rinsed with PBS three times, mounted with a vector-shield mounting medium with DAPI (#H-1500, Vector Laboratories, Newark, CA, USA), and visualized using a Leica confocal microscope.

MCL cells were treated with or without BTKi (2 μM) for 24 h, and cell lysates were prepared using Pierce™ RIPA buffer (#89900; Thermo Fisher Scientific) with Protease/Phosphatase Inhibitor Cocktail (#5872; Cell Signaling Technology) at 4 °C and quantified using Pierce™ BCA Protein Assay Kit (#23227; Thermo Fisher Scientific) before applying to a 10% polyacrylamide gel and transferring to a polyvinylidene difluoride membrane (#IPVH00010) from Immobilon-P. Antibodies against DGAT2 (#ab237613) and ENPP2 (#ab140915) were purchased from Abcam. Antibodies against SCD (#2794), ACACA (#3663), cleaved caspase-3 (#9661), cleaved PARP (#5625) and GAPDH (#5174) were all obtained from Cell Signaling Technology. Signals were detected using SuperSignal West Femto Maximum Sensitivity Substrate (#34095; Thermo Fisher Scientific) and images were acquired with Mini Chemiluminescent Imaging and Analysis System (Sage Creation Science, Beijing, China). Integrated optical density (IOD) of bands was evaluated by densitometry and analyzed using Gel-Pro Analyzer 4.0 software (Media Cybernetics, MD).