Clone 9b11

Primary antibodies used at the indicated dilutions were rabbit α-DP (used at 1:200; A303-356A; Bethyl Laboratories); mouse α-Myc (used at 1:100 for immunostainings and 1:1000 for Western blottings; clone 9B11; Cell Signaling Technology); rabbit α-γ-catenin (PG; used at 1:200 for colocalization stainings and 1:1000 for proximity ligation assay (PLA); ab15153; Abcam); rabbit α-KIAA1217 (used at 1:500 for colocalization stainings and PLA; PA5-85468; Invitrogen; kindly provided by Tobias Zech, University of Liverpool); mouse α-Desmocollin 2/3 (used at 1:200 for colocalization stainings and PLA; clone 7G6; 32-6200; Invitrogen); mouse α-p120 catenin (used at 1:400 for colocalization stainings and 1:1000 for PLA; 612536; BD Transduction Laboratories), and mouse α-Lamin A/C (used at 1:200 for colocalization stainings and PLA; mab636; MA3-1000; Invitrogen). Secondary antibodies conjugated to Alexa Fluor 488 or 647 were from Jackson ImmunoResearch and used at 1:500 for immunostainings and α-mouse Alexa Fluor 680 from Thermo Fisher Scientific was used at 1:5000 from Western blotting. Alexa Flour 680–conjugated streptavidin was used at 1:500 dilution for immunostainings and Alexa Fluor 800 was used at 1:1000 for Western blotting (both from Thermo Fisher Scientific). 4′,6-diamidino-2-phenylindole readymade solution (Sigma) was used at a concentration of 1 μg/ml for immunostainings.

L6 myocytes were incubated in serum-free alpha-MEM for 3 hours, and then stimulated with 100 nM insulin for 20 minutes if necessary. Cells were placed on ice, washed twice with ice-cold phosphate-buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4) and fixed with 3% ice-cold paraformaldehyde (PFA) on ice for 10 minutes then at room temperature for a further 10 minutes. After removing the PFA, cells were washed twice with PBS and incubated with 100 mM glycine for 10 min to quench excess PFA. Cells were washed three times with PBS and blocked with 2% BSA in PBS for 15 min. To detect surface myc-GLUT4, cells were incubated with anti-myc primary antibody (mouse monoclonal, Cell Signaling Technologies clone 9B11, 1:1000 dilution in 2% BSA in PBS) at 4 °C for 1 hour with shaking. Following five PBS washes, cells were incubated with HRP-conjugated anti-mouse secondary antibody (1:1000 dilution in 2% BSA in PBS) at 4 °C for 45 minutes with shaking. For visualisation of HRP signal (surface GLUT4 expression), cells were washed five times in PBS then incubated with 3,3′,5,5′-Tetramethylbenzidine for three to five minutes. The reaction was quenched with 2 N HCl, and the absorbance at 450 nm recorded using a plate reader. Average background signal from cells in which the primary antibody step was omitted was subtracted from each well.