Ab37977

BV2 cells were fixed with 4% paraformaldehyde for 1 h on ice, and permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature. Then, they were blocked with 5% milk at room temperature for 1 h. Next, they were incubated with primary antibodies for FAAH (#101600, 1:100, Cayman Chemical, Ann Arbor, MI, USA), SphK1 (#10670-1-AP, 1:100, Proteintech, Manchester, UK; #Sc-365401, 1:100, Santa Cruz Biotechnology, Heidelberg, Germany) or SphK2 (#ab37977, 1:50, Abcam, Cambridge, UK) for 1 h at room temperature. Thereafter, an incubation with Alexa Fluor 488-conjugated secondary antibodies (1:1000) was performed for 1 h in the dark at room temperature. Finally, the cells were washed with PBS and stained with DAPI (1 µg/mL) for 90 s in the dark at room temperature. Confocal laser scanning microscopy was performed with a Zeiss LSM510 Meta system equipped with an inverted Observer Z1 microscope and a Plan-Apochromat 63×/1.4 oil immersion objective (Carl Zeiss MicroImaging GmbH, Jena, Germany). The following excitation (ex) laser lines and emission (em) filter sets were used: DAPI: ex 405 nm, em band-pass 420–480 nm; Alexa Fluor 488: ex 488 nm, em long-pass 505 nm. Spatial calibration of the images was carried out with the “set scale” function. Microscopic images were processed with the ZEN software v.2009 (Carl Zeiss MicroImaging GmbH).

For experiments where U2OS cells were exposed to compound for less than 24 h, 60,000 cells/well were seeded onto 12-well plates. For the experiments comparing cells growing in 10% FCS/starvation for all U2OS (30,000/60,000 cells/well), MCF7 and RPE (40,000/80,000 cells/well). RIPA buffer with protease and phosphatase inhibitors (Sigma, Germany) was used for preparing protein lysates from U2OS cells treated as indicated. Immunoblotting was performed following standard protocols with indicated antibodies: ATF4 (D4B8) (1:500 or 1:50, Cell Signaling Technology, 11815S), p-P70S6K (Thr389) (1:1,000, Cell Signaling Technology, 9205), 4E-BP1 (1:1,000, Cell Signaling Tech, 9452S), p-eIF2α (phospho S51) [E90] (1:1,000, Abcam, ab32157), eIF2α (1:500, Cell Signaling Technology, 9722), Vinculin (EPR8185) (1:2,000, Abcam, ab129002), PERK (D11A8) (1:500, Cell Signaling Technology, 5683), SPHK1(D1H1L) (1:500, Cell Signaling Technology, 12071S), SPHK2 (1:50, Abcam, ab37977), and GAPDH (1:100, Abcam, ab9485). Protein bands were visualized by chemiluminescence (either with ECL, Thermo Scientific, 34076, or Amersham ECL, GE Healthcare, RPN2235) and imaged on an Amersham Imager 600 (GE Healthcare, USA). In order to detect changes in SPHK2, and ATF4 simultaneously, samples were analyzed using JESS/WES immunoblot system (ProteinSimple, USA).