Supplemental Material

This article contains the following supporting material:

• Supplementary Figure 1 - HEK293 cells were co-transfected with expression vectors for GRK6AΔ30 and either HA-tagged KDEL receptor (Golgi-restricted mutant) (Matallanas et al., 2006) or M1-mRFP (M1 protein from infectious bronchitis virus fused to mRFP) (Chiu et al., 2002). Localization of GRK6AΔ30 and the indicated organelle was visualized by immunofluorescence microscopy after co-staining with anti-GRK4-6 monoclonal antibody and anti-HA polyclonal antibody (upper panel) or anti-GRK4-6 monoclonal antibody and intrinsic mRFP fluorescence of M1-mRFP (lower panel). Images were obtained with using an Olympus BX-61 microscope and Hamamatsu ORCA-ER camera controlled by Slidebook v4.0 (Intelligent Imaging Innovations, Denver, CO). Image stacks were deconvolved using a constrained iterative algorithm in Slidebook v4.0, and an image through the middle of a cell is presented.
• Supplementary Figure 2 - HEK293 cells were transfected with expression vectors for wild type GRK6A, GRK6AΔ30 or GRK6AΔ16. Cell fractionation was performed under standard hypotonic conditions, as described under Materials and Methods, or with the addition of 0.5 M NaCl. Immunoblotting of particulate (P) and soluble (S) fractions was performed using the anti-GRK4-6 monoclonal antibody.
• Supplementary Figure 3 - HEK293 cells were transfected with expression vectors for wild type GRK6A or GRK6AΔ16. Immunofluorescent staining was performed using the anti-GRK4-6 monoclonal antibody. Images were obtained as described in Supplementary Figure 1. A maximum projection image is shown.