Figure S3.
Rap2 protein levels across Rab40 mutant cell lines and generation of Rap2 lysine mutants. (A) Rab40b binding to Rap2a, Rap2b, and Rap2c. MDA-MB-231 lysates stably expressing FLAG-Rab40b WT were incubated with GST, GST-Rap2a, GST-Rap2b, or GST-Rap2c followed by a GST pull-down assay. 25 µg of lysate input was loaded as a positive control and used to estimate pull-down efficiency. (B) Western blot showing stable overexpression of FLAG-Rab40b SOCS-4A and FLAG-Rab40b ΔSOCS in MDA-MB-231s. Ctrl cells are dox-inducible Cas9 MDA-MB-231s that were used to generate CRISPR lines. FLAG-Rab40b SOCS-4A and FLAG-Rab40b ΔSOCS were made in a Rab40b KO background. 50 µg of lysate was loaded for each sample. (C) Rap2 protein levels Ctrl versus Rab40b SOCS-4A versus Rab40b ΔSOCS cells. Ctrl, Rab40b SOCS-4A, and Rab40b ΔSOCS cell lysates were probed for αRap2 and αTubulin (loading control). Ctrl cells are dox-inducible Cas9 MDA-MB-231s that were used to generate CRISPR lines. FLAG-Rab40b SOCS-4A and FLAG-Rab40b ΔSOCS were made in a Rab40b KO background. 50 µg of lysate was loaded for each sample. (D) Quantification of Western blot in C. Three biological replicates were performed. Relative intensity of Rap2 was normalized to the levels of Tubulin and to Ctrl cells. Mean ± SD. One-way ANOVA with Tukey’s multiple comparisons test. (E) Rap2 protein levels, Ctrl versus Rab40 KO cells. Ctrl and Rab40 KO cell lysates (lacking Rab40a, Rab40b, and Rab40c) were probed for αRap2 and αTubulin (loading control). Ctrl cells are dox-inducible Cas9 MDA-MB-231s that were used to generate CRISPR lines. 50 µg of lysate was loaded for each sample. (F) Protein sequence alignment of Rap2a-WT, K5R, K3R, and K2R. Red boxes denote the five lysines within K5R, blue shades represent K2R, and green shades represent K3R. Alignment made using Clustal Omega. (G) GTP hydrolysis assay Rap2a-WT versus Rap2a-K5R. CytoPhos assay was performed using purified GST-Rap2a-WT and GST-Rap2a-K5R. Colorimetric change at 650 nm was measured and analyzed against a standard curve to determine inorganic phosphate release (nmol). Three biological replicates were performed. Mean ± SD. Unpaired t test. WT versus K5R, P = 0.0004. (H) Western blot showing stable overexpression of Eos-Rap2a-K5R and -K3R in MDA-MB-231s (lentivirus). Ctrl cells are MDA-MB-231 parentals. 50 µg of lysate was loaded for each sample. Source data are available for this figure: SourceData FS3.

Rap2 protein levels across Rab40 mutant cell lines and generation of Rap2 lysine mutants. (A) Rab40b binding to Rap2a, Rap2b, and Rap2c. MDA-MB-231 lysates stably expressing FLAG-Rab40b WT were incubated with GST, GST-Rap2a, GST-Rap2b, or GST-Rap2c followed by a GST pull-down assay. 25 µg of lysate input was loaded as a positive control and used to estimate pull-down efficiency. (B) Western blot showing stable overexpression of FLAG-Rab40b SOCS-4A and FLAG-Rab40b ΔSOCS in MDA-MB-231s. Ctrl cells are dox-inducible Cas9 MDA-MB-231s that were used to generate CRISPR lines. FLAG-Rab40b SOCS-4A and FLAG-Rab40b ΔSOCS were made in a Rab40b KO background. 50 µg of lysate was loaded for each sample. (C) Rap2 protein levels Ctrl versus Rab40b SOCS-4A versus Rab40b ΔSOCS cells. Ctrl, Rab40b SOCS-4A, and Rab40b ΔSOCS cell lysates were probed for αRap2 and αTubulin (loading control). Ctrl cells are dox-inducible Cas9 MDA-MB-231s that were used to generate CRISPR lines. FLAG-Rab40b SOCS-4A and FLAG-Rab40b ΔSOCS were made in a Rab40b KO background. 50 µg of lysate was loaded for each sample. (D) Quantification of Western blot in C. Three biological replicates were performed. Relative intensity of Rap2 was normalized to the levels of Tubulin and to Ctrl cells. Mean ± SD. One-way ANOVA with Tukey’s multiple comparisons test. (E) Rap2 protein levels, Ctrl versus Rab40 KO cells. Ctrl and Rab40 KO cell lysates (lacking Rab40a, Rab40b, and Rab40c) were probed for αRap2 and αTubulin (loading control). Ctrl cells are dox-inducible Cas9 MDA-MB-231s that were used to generate CRISPR lines. 50 µg of lysate was loaded for each sample. (F) Protein sequence alignment of Rap2a-WT, K5R, K3R, and K2R. Red boxes denote the five lysines within K5R, blue shades represent K2R, and green shades represent K3R. Alignment made using Clustal Omega. (G) GTP hydrolysis assay Rap2a-WT versus Rap2a-K5R. CytoPhos assay was performed using purified GST-Rap2a-WT and GST-Rap2a-K5R. Colorimetric change at 650 nm was measured and analyzed against a standard curve to determine inorganic phosphate release (nmol). Three biological replicates were performed. Mean ± SD. Unpaired t test. WT versus K5R, P = 0.0004. (H) Western blot showing stable overexpression of Eos-Rap2a-K5R and -K3R in MDA-MB-231s (lentivirus). Ctrl cells are MDA-MB-231 parentals. 50 µg of lysate was loaded for each sample. Source data are available for this figure: SourceData FS3.

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