Ab99532

Antibodies against OGT (ab96718), OGA (MGEA5, ab124807), O-GlcNAc (RL2, ab2739), PLIN1 (ab3526), SNAP23 (ab3340), ATGL (ab99532), DGAT1 (11561-1-AP), Fsp27 (CIDE C, ab77115), and p-Ser (phosphoserine, PSR-45) (Abcam); p492-phosphorylated PLIN1 (4855) and p517-phosphorylated PLIN1 (4856) (Vala Sciences); HA (H3663) (Sigma-Aldrich); CGI-58 (ABHD5, 12201-1-AP), PLIN2 (15294-1-AP), and PLIN3 (TIP47, 10694-1-AP) (Proteintech); p563-HSL (4139), phospho-Akt (Ser473, 9271), and Akt (9272) (Cell Signaling Technology); Myc (sc-40), DGAT2 (sc-66859), and β-actin (sc-8432) (Santa Cruz Biotechnology) were purchased from the indicated sources. Horseradish peroxidase-conjugated secondary antibodies were from Santa Cruz Biotechnology. Alexa Fluor 594-conjugated secondary antibodies, BODIPY 493/503, and BODIPY 558/568 C12 fatty acid were obtained from Thermo Fisher Scientific. 4′,6-Diamidino-2-phenylindole (DAPI), OA, Fsk, and IBMX were from Sigma-Aldrich. CL-316,243 was from R&D Systems. OGT inhibitor ST045849 (ST04) was purchased from TimTec. TMG was from Cayman Chemical.

Semi-quantitative western blotting was used to analyze the abundance of PNPLA3 in adipose, liver tissue, and primary hepatocytes. Detailed sample preparation and Western methods are provided in the Supplemental Methods. Due to apparent differences in relative PNPLA3 abundance between tissues, the primary PNPLA3 antibody (ab81874; Abcam, Cambridge, MA) was diluted to 1:250 for adipose tissue and 1:500 liver tissue and primary bovine hepatocyte samples for a 1 h room temperature incubation. After primary incubation, blots were washed and the secondary antibody (diluted 1:5000 for all samples types; ab97080, Abcam) and HRP conjugate (161-0381; Bio-Rad Laboratories, Richmond, CA) were applied for 1 h at room temperature. Blot images for total lane protein and the PNPLA3 band were captured on the ChemiDoc XRS + Imager using Image Lab 5.0 software (Bio-Rad Laboratories, Richmond, CA). The abundance of PNPLA3 protein was normalized to total lane protein within Image Lab 5.0 (Bio-Rad Laboratories, Richmond, CA) for statistical evaluation. Hepatocyte blots were also probed for ATGL abundance with the procedure detailed above, except the primary antibody (ab99532; Abcam) diluted to 1:3000 was incubated overnight at 4 °C and a different secondary antibody (ab97051, Abcam) diluted 1:5000 was used (Supplemental Methods).