Depletion of CHC22 or p115, but not GM130, sortilin, or IRAP, disrupts perinuclear targeting of GLUT4. (A) Immunoblotting for CHC22, CHC17, p115, GM130, and β-actin after transfection of HeLa-GLUT4 cells with siRNA targeting CHC17, CHC22, p115, GM130, sortilin, IRAP, or nontargeting control siRNA (siControl). The position of MW markers is indicated in kilodaltons at the left. (B) Representative IF staining for CHC22 (red) and p115 (blue) in HeLa-GLUT4 cells after siRNA transfection as in A, with GLUT4 detected by GFP tag (green). N, nuclei. Arrows point to a CHC22-depleted cell. Scale bars: 10 µm. (C) Representative IF staining for GM130 (yellow), p115 (red), and CHC22 (blue) in HeLa-GLUT4 cells after treatment with siRNA targeting GM130 or with control siRNA, with GLUT4 detected by GFP tag (green). Individual antibody staining is shown in black and white, while the merged image shows all four colors with overlap indicated as below. Scale bars: 25 µm. (D and E) Representative IF staining for GLUT4 (green), CHC22 or sortilin (red), and p115 (blue) in HeLa-GLUT4 cells after treatment with siRNA targeting sortilin or IRAP, or with nontargeting control. Scale bars: 5 µm. Merged images show red/green overlap in yellow, red/blue overlap in magenta, green/blue overlap in cyan, and red/green/blue overlap in white.