Fig 1: Intracellular localization of IMPDH2 regulates GTP levels in cell protrusions, RAC1 activity, and cell invasion.a Cells transduced with the indicated constructs (Cl sh control shRNA, IMsh IMPDH2 shRNA, IM-LCK IMPDH2 with LCK membrane-localization domain, IM-GIA IMPDH2 with giantin Golgi localization domain) were separated into the plasma membrane and cytoplasm fractions as described in Methods, followed by immunoblotting with the indicated antibodies Shown are representative images of at least two independent experiments. b Immunofluorescence analysis for IMPDH2 (red) and actin (phalloidin, green), and nuclei (Hoechst, blue) in MDA-MB-231 cells transduced with the indicated constructs. Arrows denote the presence of IMPDH2 at the cell plasma membrane. Scale bar 20 µm. Shown are representative images of two independent experiments. c GTP levels were determined via mass spectroscopy as described in Methods. The data represents average ± SEM of two independent experiments performed in duplicates. Statistics performed by two-tailed unpaired Student’s t-test (****p < 0.0001), exact p value <0.00001 for both cases. d Quantification of GEVAL activity in cell bodies (CB) and cell protrusions (CP) of MDA-MB-231 cells transduced with the indicated constructs and with GEVAL30 or GEVALNull (30 CBs and 30 CPs per cell type). Horizontal bars represent average. Individual values are from two experiments (15 CB and 15CP per experiment). Statistics were performed by a two-tailed unpaired Student’s t-test. e Cells transduced with the indicated constructs were probed in RAC1 activity assay as described in Methods. Shown are representative images of three independent experiments. f Quantification of (e). The data represents the average ± SEM of three independent experiments. Statistics were performed by a two-tailed unpaired Student’s t-test. g Invasion assay of MDA-MB-231 cells transduced with the indicated constructs. The data represents the average ± SEM of three independent experiments performed in duplicates. Statistics were performed by two-tailed unpaired Student’s t-test.
Fig 2: RAC1 interacts with IMPDH2 and recruits GMPS.a Cells were transduced with the indicated constructs and subjected to immunoprecipitation with the antibodies indicated on the top. The immunoprecipitated materials were probed in immunoblotting with the antibodies indicated on the left. Shown are representative images of at least two independent experiments. b Cells were fixed with ice-cold methanol and separated into cell bodies (CB) or cell protrusions (CP) fractions, followed by immunoblotting with the indicated antibodies. Shown are representative images of two independent experiments. c Proximity ligation assay was performed on sparsely plated cells with the antibodies indicated on the top. Shown are representative images of two independent experiments. d Schematic representation of IMPDH2 deletion mutants. Shown are Bateman domain consisting of two cystathionine-ß-synthase sequences (CBS). e Indicated GST-tagged recombinant IMPDH2 mutants were incubated with recombinant 6xHis-tagged RAC1 (shown on the top), followed by immunoprecipitation with anti-GST tag antibodies and immunoblotting with anti-RAC1 antibodies. Shown are representative images of two independent experiments. f Recombinant full-length IMPDH2 and RAC1 proteins were cross-linked followed by mass spectroscopy as described in Methods. Shown are intra-proteins cross-linked peptides identified with highest confidence. Highlighted in red are cross-linked lysines. Shown are representative gel images of at least two independent experiments.
Fig 3: Delineation of IMPDH2–RAC1 interactions.a Structures of RAC1 (shown in green) in complex with other indicated proteins (shown in blue). The number in parenthesis indicates the corresponding entry in RCSB (Research Collaboratory for Structural Bioinformatics) Protein Data Bank. Note that RAC1 binds to a-helices (shown in red) in the target proteins. b A model of the IMPDH2–RAC1 interaction was created by superimposing the a-helix of IMPDH2 containing K134 (shown in red) onto the analogous helix in the RAC1-Kalirin complex. c Convolved evolutionary coupling of IMPDH2 and RAC1. Strength of the couplings between residues of the two proteins are color-coded with red color denoting the highest probability. Shown in colored frames are regions with a high (green), intermediate (yellow), and low (blue) probability of interactions. d Cells were transduced with control shRNA (Cl sh), IMPDH2 shRNA (IMsh), or IMPDH2 shRNA in combination with wild-type IMPDH2 (IMWT), IMPDH2 lacking 153-225 aa (IM?1), or IMPDH2 lacking 101-134 aa (IM?2). Cells were subjected to immunoprecipitation with the antibodies indicated on the top. The immunoprecipitated materials were probed in immunoblotting with the antibodies indicated on the left. Shown are representative images of at least two independent experiments. e GTP levels were determined via mass spectroscopy as described in Methods. The data represents the average ± SEM of two independent experiments performed in duplicates. Statistics performed by two-tailed unpaired Student’s t-test (****p < 0.0001), exact p value < 0.00001. The data represents the average ± SEM of two independent experiments performed in duplicates. f Cells transduced with the indicated constructs were probed in RAC1 activity assay as described in Methods. Shown are representative images of at three independent experiments. g Quantification of (f). The data represents the average ± SEM of three independent experiments. Statistics were performed by a two-tailed unpaired Student’s t-test.
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