GAS2L1 CH and GAR domains form an autoinhibitory module. (A) Schematic of GAS2L1 fragments prepared for binding and sedimentation assays. (B and C) GAS2L1 constructs (FLAG-tagged) were transiently expressed in HEK293T cells for anti-FLAG immunoprecipitation. Samples of immunoprecipitates (50%) and cell-lysate inputs (1%) were immunoblotted (WB) with antibodies against FLAG and β-actin. IP, immunoprecipitation; WB, Western blotting. (D) Recombinant GAS2L1 proteins (5 µM; His6-FLAG tagged) were incubated with polymerized F-actin. After sedimentation of F-actin, pellets and supernatants were collected for anti-His6-tag and anti-β-actin immunoblotting. Sup, supernatant. To test the inhibitory effect of GAR protein, His6-FLAG-CH (5 µM) was preincubated with His6-FLAG-GAR (25 µM) for 30 min before binding with F-actin. Proportions of GAS2L1 proteins in pellets were quantified and are presented as means ± SD of three independent assays. One-way ANOVA; **, P < 0.001. (E) His6-FLAG-tagged GAS2L1 proteins (10 µM) were incubated with taxol-stabilized microtubules in the sedimentation assay, and the distribution of the proteins into the microtubule pellets and the supernatants was probed by means of anti-His6-tag and anti-α-tubulin immunoblotting. To test the effect of CH protein, His6-FLAG-CH (50 µM) was preincubated with His6-FLAG-GAR (10 µM) for 30 min. GAS2L1 proteins that cosedimented with microtubules were quantified, and the data are presented as means ± SD of three independent experiments. One-way ANOVA; **, P < 0.001. (F) GAS2L1 fragments (FLAG-tagged) were transiently coexpressed in HEK293T cells with the CH domain (GFP-tagged) for anti-FLAG immunoprecipitation. Aliquots of immunoprecipitates (50%) and cell-lysate inputs (1%) were immunoblotted (WB) with antibodies against FLAG and GFP. (G) Recombinant proteins of CH domain (His6 tagged; 2 µM) and GAR domain (His6-FLAG tagged; 1 µM) were incubated, and the mixtures were then subject to anti-FLAG immunoprecipitation. Immunoprecipitates (50%) and inputs (5%) were analyzed through anti-His6 immunoblotting.
Figure 5.

GAS2L1 CH and GAR domains form an autoinhibitory module. (A) Schematic of GAS2L1 fragments prepared for binding and sedimentation assays. (B and C) GAS2L1 constructs (FLAG-tagged) were transiently expressed in HEK293T cells for anti-FLAG immunoprecipitation. Samples of immunoprecipitates (50%) and cell-lysate inputs (1%) were immunoblotted (WB) with antibodies against FLAG and β-actin. IP, immunoprecipitation; WB, Western blotting. (D) Recombinant GAS2L1 proteins (5 µM; His6-FLAG tagged) were incubated with polymerized F-actin. After sedimentation of F-actin, pellets and supernatants were collected for anti-His6-tag and anti-β-actin immunoblotting. Sup, supernatant. To test the inhibitory effect of GAR protein, His6-FLAG-CH (5 µM) was preincubated with His6-FLAG-GAR (25 µM) for 30 min before binding with F-actin. Proportions of GAS2L1 proteins in pellets were quantified and are presented as means ± SD of three independent assays. One-way ANOVA; **, P < 0.001. (E) His6-FLAG-tagged GAS2L1 proteins (10 µM) were incubated with taxol-stabilized microtubules in the sedimentation assay, and the distribution of the proteins into the microtubule pellets and the supernatants was probed by means of anti-His6-tag and anti-α-tubulin immunoblotting. To test the effect of CH protein, His6-FLAG-CH (50 µM) was preincubated with His6-FLAG-GAR (10 µM) for 30 min. GAS2L1 proteins that cosedimented with microtubules were quantified, and the data are presented as means ± SD of three independent experiments. One-way ANOVA; **, P < 0.001. (F) GAS2L1 fragments (FLAG-tagged) were transiently coexpressed in HEK293T cells with the CH domain (GFP-tagged) for anti-FLAG immunoprecipitation. Aliquots of immunoprecipitates (50%) and cell-lysate inputs (1%) were immunoblotted (WB) with antibodies against FLAG and GFP. (G) Recombinant proteins of CH domain (His6 tagged; 2 µM) and GAR domain (His6-FLAG tagged; 1 µM) were incubated, and the mixtures were then subject to anti-FLAG immunoprecipitation. Immunoprecipitates (50%) and inputs (5%) were analyzed through anti-His6 immunoblotting.

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