Ar1 lsm confocal microscope

All images of SNAP-IDR and SNAP-hnRNPA1 were acquired on a Delta Vision epi-fluoresence microscope, equipped with a SCMOS camera. The EGFP-IDR recruitment assay was performed on a Nikon AR1 LSM confocal microscope. All images of SNAP-PTB-IDR were acquired on a Leica-based spinning disk confocal microscope (EMCCD digital camera, ImagEM X2, Hamamatsu; confocal scanner unit, CSU-X1, Yokogawa).

U-2 OS cells were grown in 96 well plates. At times indicated, media was replaced with media containing 0.5mM NaAsO₂; 200mM 2DG, 100μM CCCP or 0.5mM NaAsO₂, 200mM 2DG and 100μM CCCP for the times indicated in Figure 5. Cells were imaged using a Nikon AR1 LSM confocal microscope. For time lapse images, cells were fixed at time points given in Figure 5, as described in Buchan et al (Buchan et al., 2013 (link)) (these images were taken using the Deltavision). Fluorescence recovery after photobleaching (FRAP) experiments were done as follows: bleach areas, approximately the size of granule (~2μm), were determined. Images were taken every 5s during recovery. Mean intensity within similar sized areas was determined at each time point using ImageJ (I_t). Mean intensity within a different part of the cytoplasm was also measured (I_Bt) to correct for bleaching during image acquisition. Corrected mean intensity at each time point was determined by taking the ratio: I_t/I_Bt. Curve for exponential recovery was determined using MATLAB.