Fig. 3

Lck interacts with mitochondrial CRIF1. (a) Jurkat and Jcam whole cell lysates were immunoprecipitated (IP) with anti-CRIF1 antibody, followed by Lck and CRIF1 immunoblotting. CRIF1 immunoblot was stripped and then reblotted with anti-phosphotyrosine (pTyr) antibody. Equal amounts of Jurkat whole cell lysate were also immunoprecipitated with normal IgG as a negative control (lane 1). (b) Mitochondrial proteins isolated from Jurkat cells were immunoprecipitated with either anti-CRIF1 antibody or control IgG, and then subjected to Lck and CRIF1 immunoblotting (left panels). A fraction of mitochondrial lysate was analyzed by lamin B1 and GAPDH immunoblotting to confirm the absence of nuclear and cytosolic contamination, respectively (right panels, lane 1). Jurkat whole cell lysate was used as a positive control (right panels, lane 2). (c) Jurkat cells were subjected to immunofluorescence microscopy with three-color staining for CRIF1 (green), mito-tracker (blue), and Lck (red). Cells were also stained with DAPI to visualize nuclei (grey on upper panels). An area of three-color merged image bordered with white lines is enlarged on the right to show better resolution (lower panels). White arrows indicate co-localization of Lck and CRIF1 in mitochondria (white dots). White arrowheads depict co-localization of Lck and CRIF1 in the nucleus (yellow dots). (d) Jurkat and Jcam cells were subjected to PLA microscopy using primary antibodies specific for Lck and CRIF1 (upper panels). Green fluorescence indicates Lck and CRIF1 interaction in situ (white arrows). Secondary antibodies alone were used as negative controls (lower panels). Scale bars of 10 μm are shown in the bottom of microscopy images