High-content imaging, immunoblot and immunofluorescence data related to: Caprin-1 binding to the critical stress granule protein G3BP1 is influenced by pH

G3BP is the central node within stress-induced protein–RNA interaction networks known as stress granules (SGs). The SG-associated proteins Caprin-1 and USP10 bind mutually exclusively to the NTF2 domain of G3BP1, promoting and inhibiting SG formation, respectively. Herein, we present the crystal structure of G3BP1-NTF2 in complex with a Caprin-1-derived short linear motif (SLiM). Caprin-1 interacts with His-31 and His-62 within a third NTF2-binding site outside those covered by USP10, as confirmed using biochemical and biophysical-binding assays. Nano-differential scanning fluorimetry revealed reduced thermal stability of G3BP1-NTF2 at acidic pH. This destabilization was counterbalanced significantly better by bound USP10 than Caprin-1. The G3BP1/USP10 complex immunoprecipated from human U2OS cells was more resistant to acidic buffer washes than G3BP1/Caprin-1. Acidification of cellular condensates by approximately 0.5 units relative to the cytosol was detected by ratiometric fluorescence analysis of pHluorin2 fused to G3BP1. Cells expressing a Caprin-1/FGDF chimera with higher G3BP1-binding affinity had reduced Caprin-1 levels and slightly reduced condensate sizes. This unexpected finding may suggest that binding of the USP10-derived SLiM to NTF2 reduces the propensity of G3BP1 to enter condensates.
Methods
The deposited ZIP folders are described in following groups:

@HTimages_1

high-throughput imaging datasets of cellular GFP-G3BP condensation assays presented in Figure 5 of the manuscript
ZIP folders: img_plate2171.zip / img_plate2185.zip / img_plate21691.zip

@HTimages_2

high-throughput imaging datasets of cellular GFP-Caprin condensation assays presented in Figure 7 of the manuscript.
ZIP folders: img_plate1734.zip / img_plate1735.zip / img_plate1737.zip / img_plate1740.zip

@HTimages_3

high-throughput imaging datasets of in-vitro reconstituted GFP-G3BP condensation assays (LLPS), presented in Figure 5 of the manuscript.
ZIP folders: LLPS_GFP-G3BP1.zip

@HTimages_4

high-throughput imaging datasets of in-vitro reconstituted GFP-Caprin condensation assays (LLPS), presented in Figure 7 of the manuscript.
ZIP folders: LLPS_GFP-Caprin-1.zip

@CPpipelines_1

Cellprofiler pipelines to analyse @HTimages_1 
ZIP folders: pipelines_G3BP1.zip t

@CPpipelines_2

Cellprofiler pipelines to analyse @HTimages_2 
ZIP folders:pipelines_Caprin1.zip

@Images_1

raw image files used for presentation of Blots or IF data in main and supplemental figures
ZIP folders: Images_Blots.zip

The collection of datasets is described in the methods section of the associated manuscript (ms, https://royalsocietypublishing.org/doi/10.1098/rsob.220369)

@HTimages_1/@HTimages_2:

ms section: 4.9.1. High-content microscopy: 
"Images were recorded with a Molecular Devices ImageXpress Micro microscope, equipped with a 20x or 40x objective, and illuminated with a mercury lamp and standard filters for DAPI, FITC, Cy3 and Cy5. Images were captured using a four-megapixel sCMOS digital camera with the manufacturer’s software MetaXpress, and raw TIF files were analyzed using CellProfiler (CP), ImageJ and Rstudio."

@HTimages_3/@HTimages_4

ms section: 4.9.4. In vitro reconstituted condensate assays
"Images were taken with a Supercontinuum Confocal Leica TCS SP5 X, equipped with a pulsed white light laser and a Leica HCX PL Apo 63x/1.40 oil objective. "

@Images_1

ms section 4.8. Immunoprecipitation and immunoblotting

ms section: 4.9. Immunofluorescence analysis