Anti dgkζ

Antibodies to p-S6K Thr389 (9205), S6K (9202), p-AKT Ser473 (9271), AKT (2920), p-S6 Ser240/4 (2215), S6 (2317), p-PKD Ser744/8 (2054) and p-PKC substrate (2261) were from Cell Signaling (Danvers, MA, USA), anti-PKCα Ser657 from US Biologicals (Swappscott, MA, USA) (P9103-16A), anti-PKCα and PKD from Santa Cruz Biotechnology (Santa Cruz, CA, USA) (sc-208, -935, -935), anti-SREBP-1 from Millipore (Temecula, CA, USA) (04-469), anti-DGKζ from Abcam (Cambridge, UK) (ab105195), anti-DGKα from Abnova (Tapei, Taiwan) (BO1P), anti-tubulin DM1A (T9026) from Sigma-Aldrich (Saint Louis, MO, USA) and anti-phosphotyrosine (4G10) from Upstate (Lake Placid, NY, USA). Horseradish peroxidase-coupled polyclonal goat anti-mouse and -rabbit immunoglobulins were from DakoCytomation (Glostrup, Denmark) (P0447, P0448). DGK inhibitor II (R59949; 266788), bisindolylmaleimide and Gö6976 were from Calbiochem (Darmstadt, Germany), (203291, 36520). Rapamycin was from LC Laboratory (Woburn, MA, USA) (R-5000). Torin was from Tocris (Bristol, UK) (4247). Recombinant DGK from Escherichia coli (D3065), PMSF, leupeptin and aprotinin were from Sigma-Aldrich. Adenosine 5′triphosphate [γ³²P] was from Hartman Analytic (Braunschweig, Germany). All lipids were purchased from Avanti Polar Lipids (Alavaster, AL, USA). Organic reagents were from Merck (Darmstadt, Germany).

Following antibodies were used at a dilution of 1:1000 except where otherwise stated: Anti-Actin (sc-1616 HRP; 1:10,000), anti-ATP7A (sc-376467; 1:100), anti-normal mouse IgG (sc-2025; 1.4 µg for IP), anti-ERK (sc-514302) from Santa Cruz; anti-GFP (2555), anti-OTULIN (14127; 1.4 µg for IP), anti-IκBα (4814), anti-phospho-IκBα (Ser32/36) (9246), anti-LAMP1 (15665; 1:100), anti-phospho-ERK (Thr202/Tyr204) (9101), anti-normal rabbit IgG (2729; 1.4 µg for IP), anti-EEA1 (3288, 1:100) all CST; anti-SNX27 (ab77799; 1.4 µg for IP and 1:50 for IF), anti-VPS26 (ab23892), anti-VPS35 (ab157220; 1:10,000), anti-MRP4 (ab15602; 1:2500), anti-DGKζ (ab105195; 1:100), anti-GLUT1 (ab15309; 1:100 and ab115730) all Abcam; anti-Beta PIX (07-1450-I; 1:5000), anti-Met1-Ub (MABS199) all Millipore; anti-STEAP3 (17186-1-AP), anti-KIDINS220 (21856-1-AP) all Proteintech; anti-SNX27 (for murine SNX27) gift from W. Hong; anti-Beta-catenin (610153, 1:2000) BD; anti-FLAG M2 (F3165; 1:10000 and 1 µg for IP) Sigma; anti-HOIP (MAB8039; 1:5000) R&D; anti-HA (Core facility monoclonal antibodies HMGU; 25 µl for IP); anti-CD2-APC (17-0029-41; 1:200) eBioscience.