Ab109263

Intestinal tumor tissue was adequately homogenized on ice in a mixture of RIPA, PMSF, and protease inhibitors. Centrifugation was spun at 13 000 rpm to collect the supernatant containing total protein. Protein concentration was measured using detergent compatible protein assay (BIO‐RAD, Hercules, CA). Then, 40 mg of protein was separated by 5% upper gel and 12% lower gel and transferred onto polyvinylidene difluoride membranes (GE Healthcare, Piscataway, NJ). The primary anti‐β‐catenin (ab32572, Abcam,1:5000), ZO‐1 (33‐9100; Thermo Fisher Scientific, 1:1000), Claudin‐3 (34‐1700; Thermo Fisher Scientific, 1:1000), Occludin (PA5‐30230; Thermo Fisher Scientific, 1:1000), PCNA (13 110; Cell Signaling, 1:2000), Cyclin D1 (2922; Cell Signaling, 1:1000), PCYT2 (A15309, ABclonal, 1:1000) and PCYT1α (ab109263, Abcam, 1:1000) were applied. Secondary antibodies were horseradish peroxidase conjugated anti‐rabbit or anti‐mouse. Chemiluminescence signals were detected by the ECL detection kit. The intensity of Western blotting images was determined by Image J.

Protein extraction from maternal liver and immunoblotting were performed as in [28 (link)] using primary antibodies specific for MTHFD1 [41 (link)], methyleneTHF reductase (MTHFR) [42 (link)], methionine synthase (MTR) (Proteintech 25896-1-AP; ThermoFisher, Waltham, MA, USA), betaine-homocysteine methyltransferase (BHMT) [43 (link)], phosphate cytidylyltransferase 1A, choline (PCYT1A) (ab109263, Abcam), choline kinase alpha (CHKA) (Invitrogen PA5-109529, ThermoFisher), phosphatidylethanolamine N-methyltransferase (PEMT) (Invitrogen PA5-42383; ThermoFisher), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2118, Cell Signaling Technology, Danvers, MA, USA, VINCULIN (13901, Cell Signaling Technology), and ACTIN (A2066; Sigma-Aldrich, St. Louis, MO, USA). The extraction buffer contained protease inhibitors (Pierce, ThermoFisher) as well as phosphatase inhibitors (ThermoFisher) to preserve protein phosphorylation. Imaging and quantification of the blots was done using the Amersham Imager 600 (GE Healthcare Life Sciences, Marlborough, MA, USA) analysis software v1.0.0.

Cells were fixed with 4% paraformaldehyde for 15 min and permeabilised with 0.5% TritonX-100 for 10 min prior to blocking with 3% BSA for 1 h at room temperature (RT). Immunostaining with PCYT1A primary antibody (ab109263, Abcam) at 1:100 dilution was performed at 4°C overnight and was followed by washing with PBS and incubation with Alexa Fluor 594 or 647-conjugated secondary antibodies at 1:1000 dilution for 1 hour at RT. After washing with PBS, lipid droplets were stained with BODIPY 493/503 at 1:1000 dilution for 30 min at RT. Cells were mounted on microscope slides with ProLong Gold Antifade Mountant with DAPI.