Anti sx4

Anti-pan Akt (#2920), anti-phospho S473 Akt (#4058), anti-ERK1/2 (#9102), anti-phospho ERK1/2 (#9106) were from Cell Signalling (Danvers, Massachusetts, USA). Anti-Sx4 (#110042) and Anti-SNAP23 (#111202) were from Synaptic Systems (Goettingen, Germany). Anti-GLUT1 (#652) and anti-GLUT4 (654) were from AbCam (Cambridge, United Kingdom) and anti-GAPDH (#4300) was from Ambion (Foster city, California, USA). Detection antibodies were from LI-COR Biosciences (Lincoln, Nebraska, USA).

Samples were thawed and combined with Laemmli sample buffer and heated to 65°C for 10 min prior to SDS‐PAGE and immunoblotting. Blots were visualized using LICOR infra‐red fluorescence detection and quantified using company proprietary software. The antibodies used in this study were anti‐GLUT4 (Thermo Fisher Scientific Cat# PA1‐1065, RRID:AB_2191454), anti‐Sx16 (Synaptic Systems Cat# 110162, RRID:AB_887799), anti‐Sx4 (Synaptic Systems Cat# 110042, RRID:AB_887853), anti‐SNX1 (Proteintech Cat# 10304‐1‐AP, RRID:AB_2192217; the band at 70 kDa present in both 3 T3‐L1 adipocytes and skeletal muscle was quantified), anti‐SNX27 (Proteintech Cat# 16329‐1‐AP, RRID:AB_10888628), anti‐VPS35 (Proteintech Cat# 10236‐1‐AP, RRID:AB_2215216) and anti‐MAPK (Santa Cruz Biotechnology Cat# sc‐514,302, RRID:AB_2571739).

Anti-Sx4 (#110042), anti-Sx2 (#110022), anti-Sx3 (#110032), anti-Sx6 (#110062) and anti-Sx16 (#110161) were from Synaptic Systems (Göttingen, Germany) and were all used at 1:1000. Anti-GLUT1 (#652) and anti-GLUT4 (#654) were from Abcam (Cambridge, UK) (both used at 1:1000) and anti-GAPDH (#4300; used at 1:80,000) was from Ambion (Foster City, CA, USA). Detection antibodies were from LI-COR Biosciences (Lincoln, NE, USA) and were used at 1:10,000. Anti-sortilin (#12369-1-AP; 1:1000), anti-tankyrase (#18030-1-AP; 1:250) and anti-USP25 (#12199-1-AP; 1:1000) were from Proteintech. Anti-IRAP was from Paul Pilch (Boston College, Boston, MA, USA; 1:500). Anti-ACRP30 was as described previously (Clarke et al., 2006 (link); 1:1000).