The co-recruitment assay was done as previously described (48 (link)). In situ proximity ligation assay (PLA) was performed as recommended by the manufacturer (DuolinkII kit, Olink Bioscience AB). Briefly, HeLa cells grown on coverslips were fixed in PBS 1X/paraformaldehyde 4% and permeabilized with a PBS 1X/ Triton X100 0.1% solution. Primary antibodies were diluted in 1x antibody diluent and incubated for 1 h at room temperature. The negative controls used only one of each primary antibody. Cells were washed twice for 5 min in PBS 1X. The PLA probes (Rabbit-MINUS and Mouse-PLUS) were incubated in a pre-heated humidity chamber for 1 h at 37°C. Subsequent steps were performed using the detection reagents green according to DuolinkII kit protocol. The Duolink mounting medium was supplemented with 10 μM TO-PRO-3 final to counterstain for nuclei. Laser confocal microscopy was performed with a SP5-AOBS X Leica confocal microscope. Images from each channel were recorded separately and then merged. Images were processed and assembled with Photoshop CS5 (Adobe). The antibodies used in these experiments were as follows: anti-SMN (2B1), anti-Gemin2 (2E17), anti-Gemin3 (44 ), anti-Gemin4 (45 (link)), anti-Gemin6 (12307–2-AP, PTG Lab), anti-Gemin7 (6E2, Millipore), anti-Gemin8 (1F8) (20 (link)), anti-NUFIP (12515–1-AP, Proteintech) and anti-GAPDH (Abcam).
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