Journal: Scientific reports

Article Title: CD147 regulates the Rap1 signaling pathway to promote proliferation, migration, and invasion, and inhibit apoptosis in colorectal cancer cells.

doi: 10.1038/s41598-025-98266-8

Figure Lengend Snippet: Fig. 5. Rap1 overexpression reverses the expression of key proteins in CD147 knockdown mediated cell proliferation, apoptosis and EMT. (A, B) Western blot analysis of Rap1 and Rap1GAP protein levels in HCT116 and SW620 cells after CD147 knockdown (shCD147). β-actin served as a loading control. Data represent mean ± SD (n = 3; **P < 0.01, ***P < 0.001 vs. shNC, Student’s t-test). Original blots are presented in Supplementary Fig. 3. (C) qRT-PCR validation of Rap1 mRNA overexpression in CD147-knockdown cells. Expression normalized to β-actin (n = 3; ***P < 0.001 vs. HCT116 or SW620, Student’s t-test). (D, E) Western blot analysis of c-Myc, Bcl-2, Bax, N-cadherin, and E-cadherin protein levels in CD147-Knockdown cells with Rap1 overexpression (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001 vs. shNC, #P < 0.05, ##P < 0.01, ###P < 0.001 vs. shCD147, one-way ANOVA). Original blots are presented in Supplementary Fig. 4.

Article Snippet: Supplier Antibody dilution used WB/IF Anti CD147 mouse monoclonal antibody 66443-1-AP ProteinTech Group, Inc. 1:5000 /1:200 Anti N-cadherin rabbit polyclonal antibody 22018-1-AP ProteinTech Group, Inc. 1:5000 Anti E-cadherin rabbit polyclonal antibody 20874-1-AP ProteinTech Group, Inc. 1:10000 Anti c-myc mouse monoclonal antibody 67,447-AP ProteinTech Group, Inc. 1:5000 Anti RAP1GAP rabbit polyclonal antibody 19174-1-AP ProteinTech Group, Inc. 1:2000 Anti RAP1 rabbit polyclonal antibody 14595-1-AP ProteinTech Group, Inc. 1:1000 Anti β-actin rabbit polyclonal antibody 20536-1-AP ProteinTech Group, Inc. 1:8000 HRP-conjugated Goat Anti-Mouse IgG(H + L) SA00001-1 ProteinTech Group, Inc. 1:10000 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) ab150116 Abcam 1:200 Table 1.

Techniques: Over Expression, Expressing, Knockdown, Western Blot, Control, Quantitative RT-PCR, Biomarker Discovery

Journal: Scientific reports

Article Title: CD147 regulates the Rap1 signaling pathway to promote proliferation, migration, and invasion, and inhibit apoptosis in colorectal cancer cells.

doi: 10.1038/s41598-025-98266-8

Figure Lengend Snippet: Fig. 7. Rap1 restores CD147-mediated migration and invasion in colorectal cancer cells. (A, C) Scratch wound healing assay in HCT116 cells. Healing rates were quantified at 24 h and 48 h (n = 3; ***P < 0.001 vs. shNC, ##P < 0.01, ###P < 0.001 vs. shCD147, one-way ANOVA). (B, D) Transwell migration and Matrigel invasion assays. Migrated/invaded cells were counted and normalized to shCtrl (n = 3; ***P < 0.001 vs. shNC, ###P < 0.001 vs. shCD147, one-way ANOVA). SW620 cells showed consistent trends. (E) Mechanistic diagram of CD147 promoting tumor progression through Rap1/Rap1GAP signaling in colorectal cancer cells.

Article Snippet: Supplier Antibody dilution used WB/IF Anti CD147 mouse monoclonal antibody 66443-1-AP ProteinTech Group, Inc. 1:5000 /1:200 Anti N-cadherin rabbit polyclonal antibody 22018-1-AP ProteinTech Group, Inc. 1:5000 Anti E-cadherin rabbit polyclonal antibody 20874-1-AP ProteinTech Group, Inc. 1:10000 Anti c-myc mouse monoclonal antibody 67,447-AP ProteinTech Group, Inc. 1:5000 Anti RAP1GAP rabbit polyclonal antibody 19174-1-AP ProteinTech Group, Inc. 1:2000 Anti RAP1 rabbit polyclonal antibody 14595-1-AP ProteinTech Group, Inc. 1:1000 Anti β-actin rabbit polyclonal antibody 20536-1-AP ProteinTech Group, Inc. 1:8000 HRP-conjugated Goat Anti-Mouse IgG(H + L) SA00001-1 ProteinTech Group, Inc. 1:10000 Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) ab150116 Abcam 1:200 Table 1.

Techniques: Migration, Wound Healing Assay

Journal: Molecular and Cellular Biology

Article Title: TIN2-Tethered TPP1 Recruits Human Telomerase to Telomeres In Vivo

doi: 10.1128/mcb.00240-10

Figure Lengend Snippet: FIG. 6. The TPP1 OB-fold is required to rescue telomerase recruitment to telomeres. An shRNA-resistant form of TPP1 is able to restore hTR localization to telomeres in TPP1-depleted cells. However, an shRNA-resistant form of TPP1 lacking the OB-fold cannot restore localization. (A) Parental and TPP1-depleted super-telomerase HeLa cells were subjected to FISH and IF to detect hTR (red), coilin (blue), and TRF2 (green). Merge panels show superimposition of hTR, coilin, and TRF2. Next, parental cells were cotransfected with shTPP1 and either TPP1* or TPP1OB*. Treated cells were subjected to FISH and IF to detect hTR (red), FLAG (blue), and RAP1 (telomere marker, green). Merge panels show superimposition of hTR, FLAG, and RAP1. (B) Plot of the average number of telomere-associated hTR foci per cell in the parental cells and each experimental group. Error bars indicate standard errors calculated with N equal to the number of samples quantitated.

Article Snippet: Next, cells were incubated with one of several combinations of the following primary antibodies at the indicated dilution for 1 h at room temperature: mouse anti-p80 coilin (1:5,000, ) (1), mouse anti-TRF2 (1:1,000; Imgenex Corp., San Diego, CA), rabbit anti-hTERT (1:400; Rockland, Gilbertsville, PA), mouse anti-FLAG (1:500; Sigma-Aldrich, St. Louis, MO), rabbit anti-RAP1(1: 2,000; Novus Biologicals, Littleton, CO), and rabbit anti-53BP1(1:500; Bethyl, Montgomery, TX).

Techniques: shRNA, Marker

Journal: Journal of Cell Science

Article Title: Talin, a Rap1 effector for integrin activation at the plasma membrane, also promotes Rap1 activity by disrupting sequestration of Rap1 by SHANK3

doi: 10.1242/jcs.263595

Figure Lengend Snippet: Optogenetic recruitment of talin to the plasma membrane of endothelial cells leads to activation of Rap1. (A) Schematic representation of the optogenetic constructs CIBN–GFP–CAAX and CRY2–mCherry–talin expressed in immortalized mouse lung endothelial cells. The CIBN moiety is anchored to the plasma membrane and recruits CRY2–mCherry–talin upon exposure of the cells to 450 nm (blue) light. Previous work has shown that such recruitment leads to activation of integrin αVβ3 . Rap1 activation, the transition from Rap1–GDP to Rap1–GTP, can also be monitored during this process. (B) Time course of Rap1 activation in endothelial cells in response to blue light illumination. Rap1–GTP was selectively pulled down using agarose beads loaded with the Rap-binding domain of RalGDS and detected using an anti-Rap1 antibody. Upper panel: representative western blots of the Rap1–GTP pull-down assay and total Rap1 in whole-cell lysates (input: 5%). Lower panel: quantitative analysis of Rap1–GTP. The ratio of Rap1–GTP to total Rap1 was calculated and normalized to that observed at time zero when the cells were maintained in the dark. Data represent means±s.e.m. of four experiments (* P <0.05; paired two-tailed Student's t -test). (C) Time course of Rap1 activation in response to blue light in A5 CHO cells stably expressing integrin αIIbβ3, CIBN–GFP–CAAX and CRY2–mCherry–talin. Data represent means±s.e.m. of four experiments (* P <0.05; ** P <0.01; paired two-tailed Student's t -test). (D) Recruitment to the plasma membrane of a CRY2–mCherry–talin mutant (R118E) that cannot interact with Rap1 fails to activate Rap1 in A5 CHO cells. Data represent means±s.e.m. of four experiments (N.S., not significant; ** P <0.01; paired two-tailed Student's t -test). (E) Expression of a CRY2–mCherry–talin mutant (L325R) defective in activating integrins still enables Rap1 activation in these cells upon recruitment of CRY2–mCherry–talin to the plasma membrane in A5 CHO cells. Data represent means±s.e.m. of four experiments (* P <0.05; paired two-tailed Student's t -test).

Article Snippet: The mouse monoclonal antibody to Rap1 was from Proteintech (Rosemont, IL, USA; 67174).

Techniques: Clinical Proteomics, Membrane, Activation Assay, Construct, Binding Assay, Western Blot, Pull Down Assay, Two Tailed Test, Stable Transfection, Expressing, Mutagenesis

Journal: Journal of Cell Science

Article Title: Talin, a Rap1 effector for integrin activation at the plasma membrane, also promotes Rap1 activity by disrupting sequestration of Rap1 by SHANK3

doi: 10.1242/jcs.263595

Figure Lengend Snippet: Optogenetic recruitment of talin to the plasma membrane promotes active Rap1 localization to cell edges. (A) Optogenetic recruitment of talin to the plasma membrane promotes active Rap1 localization to the cell periphery in suspended cells. Immortalized murine endothelial cells in suspension expressing CIBN–GFP–CAAX and CRY2–mCherry–talin were illuminated using blue light for 30 min before Rap1–GTP was detected in situ as described in the Materials and Methods. Samples without GST–RalGDS were used as a control (column 1). White arrows indicate enrichment of active Rap1 and CRY2–mCherry–talin at the cell periphery, the former only in response to blue light. Scale bar: 10 µm. (B) Optogenetic recruitment of talin to the plasma membrane enriches active Rap1 localization at cell protrusions in adherent cells. Immortalized murine endothelial cells expressing CIBN–GFP–CAAX and CRY2–mCherry–talin were plated on fibronectin-coated coverslips for 30 min before being illuminated with blue light or maintained in the dark for 30 min. Samples were fixed and in situ Rap1–GTP assay was performed as described in the Materials and Methods. Samples without GST–RalGDS incubation were used as a negative control and are shown in column 1. In these representative images, cell protrusions are highlighted by the small box in the main panel and presented as magnified insets on the bottom right of each image. Blue light illumination induces active Rap1 localization on cell lamellipodium-like protrusions (column 4) and pseudopodium-like protrusions (column 5). Note that such signal enrichment was not seen in cells maintained in the dark (columns 2 and 3) despite the formation of cell protrusions. Scale bars: 20 µm (main panel); 1 µm (inset). Images in A,B are representative of three independent experiments.

Article Snippet: The mouse monoclonal antibody to Rap1 was from Proteintech (Rosemont, IL, USA; 67174).

Techniques: Clinical Proteomics, Membrane, Suspension, Expressing, In Situ, Control, Incubation, Negative Control

Journal: Journal of Cell Science

Article Title: Talin, a Rap1 effector for integrin activation at the plasma membrane, also promotes Rap1 activity by disrupting sequestration of Rap1 by SHANK3

doi: 10.1242/jcs.263595

Figure Lengend Snippet: Overexpression of SHANK3 blocks Rap1 activation induced by talin recruitment to the plasma membrane. (A–C) SHANK3 tagged with Myc–mAzurite was transfected into A5 CHO cells stably expressing CIBN–GFP–CAAX and CRY2–mCherry–talin. Cells triple positive for mAzurite, GFP and mCherry were sorted by flow cytometry. Cells expressing Myc–mAzurite without SHANK3 served as a control. (A) Western blot analysis of SHANK3–Myc–mAzurite expression in these cells. β-actin served as a loading control. SHANK3 overexpression did not affect the levels of CRY2–mCherry–talin. The images represent two independent experiments. (B) SHANK3 overexpression inhibits Rap1 activation in response to the optogenetic recruitment of talin to the plasma membrane. Data represent means±s.e.m. of five experiments (N.S., not significant; ** P <0.01; paired two-tailed Student's t -test). (C) SHANK3 blunts activation of integrin αIIbβ3 in response to the optogenetic recruitment of talin to the plasma membrane. Activation of integrin αIIbβ3 was monitored by flow cytometry using the PAC-1 antibody and expressed as the fold increase relative to that observed when cells were maintained in the dark. Data represent means±s.e.m. of five experiments (* P <0.05; paired two-tailed Student's t -test). (D–G) Lentiviruses encoding the WT SPN domain of SHANK3 [mAzurite–FLAG–SPN (WT)], the R12C SPN mutant [mAzurite–FLAG–SPN (R12C)] or the L68P SPN mutant [Myc–mAzurite–FLAG–SPN (L68P)] were transduced into immortalized murine lung endothelial cells expressing CIBN–GFP–CAAX and CRY2–mCherry–talin. Cells infected with empty lentiviral vector served as controls. (D) WT SPN, but not R12C or L68P SPN, inhibits Rap1 activation following optogenetic recruitment of talin to the plasma membrane. Data represent mean±s.e.m. of four experiments (N.S., not significant; * P <0.05; paired two-tailed Student's t -test). (E) WT SPN, but not the R12C or L68P SPN mutants, inhibits specific fibrinogen binding to integrin αVβ3 upon optogenetic recruitment of talin to the plasma membrane. Data represent means±s.e.m. of eight experiments (N.S., not significant; * P <0.05; ** P <0.01; paired two-tailed Student's t -test). (F,G) Duolink proximity ligation assay (PLA) was performed to examine the effects of SHANK3 SPN on the association of Rap1 with CRY2–mCherry–talin in endothelial cells. (F) Schematic representation of the Duolink PLA. Created in BioRender by Liao, Z., 2025. https://BioRender.com/m47c469 . This figure was sublicensed under CC-BY 4.0 terms. (G) After 30 min of incubation at room temperature in the absence or presence of blue light illumination, cells were fixed, permeabilized and stained with rabbit anti-mCherry and mouse anti-Rap1 antibodies. Then, Duolink PLA flow cytometry was performed to assess the interaction between CRY2–mCherry–talin and Rap1. Cells kept in the dark and untreated with primary antibodies served as controls. Data represent means±s.e.m. of four experiments (* P <0.05; paired two-tailed Student's t -test).

Article Snippet: The mouse monoclonal antibody to Rap1 was from Proteintech (Rosemont, IL, USA; 67174).

Techniques: Over Expression, Activation Assay, Clinical Proteomics, Membrane, Transfection, Stable Transfection, Expressing, Flow Cytometry, Control, Western Blot, Two Tailed Test, Mutagenesis, Infection, Plasmid Preparation, Binding Assay, Proximity Ligation Assay, Incubation, Staining

Journal: Journal of Cell Science

Article Title: Talin, a Rap1 effector for integrin activation at the plasma membrane, also promotes Rap1 activity by disrupting sequestration of Rap1 by SHANK3

doi: 10.1242/jcs.263595

Figure Lengend Snippet: Optogenetic recruitment of talin to the plasma membrane impairs Rap1 interaction with SHANK3. (A) Schematic representation of the Duolink proximity ligation assay (PLA). Cells were fixed, permeabilized and stained with rabbit anti-SHANK3 and mouse anti-Rap1 antibodies before Duolink PLA was performed to assess the proximity of SHANK3 to Rap1. Created in BioRender by Liao, Z., 2025. https://BioRender.com/b40n281 . This figure was sublicensed under CC-BY 4.0 terms. (B) Immortalized murine lung endothelial cells expressing CRY2–mCherry–talin and CIBN–GFP–CAAX were plated on fibrinogen overnight, fixed, permeabilized and stained with anti-SHANK3 and anti-Rap1 antibodies. PLA was performed to evaluate colocalization of endogenous SHANK3 and Rap1. Cell nuclei were counterstained with DAPI and cells were imaged by confocal microscopy. Cells kept in the dark and untreated with primary antibodies served as controls. Two areas within the images with merged signals for PLA and CIBN–GFP–CAAX are highlighted with boxes and presented as magnified insets on the bottom. PLA signals were observed within the cytoplasm (inset on the left) and on the plasma membrane (inset on the right). Scale bars: 35 µm (main panel); 10 µm (inset). Images represent two independent experiments. (C) Immortalized murine lung endothelial cells in suspension were either kept in the dark or exposed to blue light illumination for the indicated times, before being subjected to Duolink PLA assay and analyzed by flow cytometry to quantitatively assess the interaction of endogenous SHANK3 and Rap1. Cells kept in the dark and untreated with primary antibodies served as controls. Data represent means±s.e.m. of five experiments (N.S., not significant; * P <0.05; paired two-tailed Student's t -test). Cells transduced with lentivirus encoding shRNA to knock down SHANK3 were also used as a further control to demonstrate the specificity of the PLA signal in four out of the five experiments (* P <0.05; unpaired two-tailed Student's t -test).

Article Snippet: The mouse monoclonal antibody to Rap1 was from Proteintech (Rosemont, IL, USA; 67174).

Techniques: Clinical Proteomics, Membrane, Proximity Ligation Assay, Staining, Expressing, Confocal Microscopy, Suspension, Flow Cytometry, Two Tailed Test, Transduction, shRNA, Knockdown, Control

Journal: Science signaling

Article Title: Mechanisms of autoregulation of C3G, activator of the GTPase Rap1, and its catalytic deregulation in lymphomas.

doi: 10.1126/scisignal.abb7075

Figure Lengend Snippet: Fig. 4. Mutations that disrupt the AIR/Cdc25HD interaction activate C3G constitutively. (A) Conservation scores of C3G per residue (colored bars) and average values per domain (boxes). Alignment of the sequences of the P3 motif and part of the AIR from representative species; positions are colored according to their conservation. Secondary structure predictions by three methods are shown under the sequences. Secondary structure prediction for the com- plete SH3b domain is shown in fig. S2. (B and C) Binding of the Cdc25HD to GST-AIR, WT, and mutants, analyzed by PD assays. Cdc25HD in the PD was detected by Western blot. Two types of substitutions were assayed: reverse-charge replacements (B) and changes to alanine and mutations described in lymphomas (C). PDs are representative of two independent experiments. (D) Helical wheel representation of the predicted helix in the AIR-CBR. Met551, Tyr554, and Met555 define the putative binding site for the Cdc25HD. (E and F) Representative nucleotide exchange reactions of Rap1:mant-dGDP catalyzed by full-length C3G WT and the indicated point mutants (1 M) (E) and the exchange rates (kobs) (F). n = 3 to 5 independent experiments, as indicated, one shown in (E). (G) Schematic representation of auto inhibited C3G and the uninhibited conformation induced by mutations that destabilize the AIR/Cdc25HD interaction.

Article Snippet: 13, eabb7075 (2020) 1 September 2020 13 of 17 rabbit pAb 121 against Rap1 (sc-65, Santa Cruz Biotechnology), and mouse mAb against -actin (dilution 1/2000; AC-15, SigmaAldrich).

Techniques: Residue, Binding Assay, Western Blot

Journal: Science signaling

Article Title: Mechanisms of autoregulation of C3G, activator of the GTPase Rap1, and its catalytic deregulation in lymphomas.

doi: 10.1126/scisignal.abb7075

Figure Lengend Snippet: Fig. 5. Lymphoma-related mutations in C3G activate Rap1 and LFA-1 in cells. (A) Analysis of Rap1-GTP in HEK293T cells expressing mEGFP; C3G-mEGFP WT; the mutants M551R, Y554H, or M555K; or the membrane-targeted WT with a CAAX sequence. Expression of mEGFP and endogenous Rap1 and -actin in cell lysates were analyzed by Western blot. In the PD, Rap1-GTP was detected by Western blot, and GST-RalGDS-RBD was detected by Ponceau S staining. PD is representative of three independent experiments. (B) Scatterplot and bar chart of three independent measurements of Rap1 activation in HEK293T cells, as described and represented in (A). Rap1-GTP levels in cells expressing C3G-mEGFP-CAAX were used to normalize the data from different experiments. (C) Correlation between the exchange activity in vitro (kobs) of C3G WT and mutants (shown in Fig. 4F) and the Rap1-GTP levels that they induced in HEK293T cells [shown in (B)]. (D) Rap1-GTP levels in Ba/F3 cells expressing C3G-mEGFP, WT or the indicated mutants, or mEGFP alone and in uninfected cells. Proteins were detected in the cell lysates and in the GST-RalGDS- RBD PDs as described in (A). In addition, C3G (endogenous and mEGFP-tagged) was also detected using an antibody against C3G. PD is representative of two independent experiments. (E) Time course activation of Rap1 after stimulation with IL-3 of Ba/F3 cells expressing mEGFP, C3G-mEGFP WT, or Y554H. Rap1-GTP was detected as described in (A) and (D). PD is representative of two independent experiments. (F) Activation of the integrin LFA-1 in Ba/F3 cells expressing mEGFP, C3G-mEGFP WT, or mutants, determined by flow cytometry (n = 3, biological replicates, means ± SD). Statistical comparison to cells expressing mEGFP was analyzed using ANOVA followed by Dunnett’s test. ***P < 0.001; ****P < 0.0001.

Article Snippet: 13, eabb7075 (2020) 1 September 2020 13 of 17 rabbit pAb 121 against Rap1 (sc-65, Santa Cruz Biotechnology), and mouse mAb against -actin (dilution 1/2000; AC-15, SigmaAldrich).

Techniques: Expressing, Membrane, Sequencing, Western Blot, Staining, Activation Assay, Activity Assay, In Vitro, Flow Cytometry, Comparison

Journal: Science signaling

Article Title: Mechanisms of autoregulation of C3G, activator of the GTPase Rap1, and its catalytic deregulation in lymphomas.

doi: 10.1126/scisignal.abb7075

Figure Lengend Snippet: Fig. 6. The AIR/Cdc25HD interaction is the main autoinhibitory mechanism and is disrupted by CrkL for activation. (A) Phosphorylation of purified C3G-WT with SrcKD, analyzed by Western blot. Similar analysis of Cdc25HD and the C3G mutants M551R, Y554R, and Y554H are shown in fig. S3 (A to D). (B) Representative dissociation reactions of Rap1:mant-dGDP catalyzed by C3G (1 M), unmodified or phosphorylated with SrcKD (p-C3G), alone and in the presence of CrkL (10 M). (C) Nucleotide ex- change rates (kobs) catalyzed by full-length C3G WT and point mutants and the isolated Cdc25HD. Unphosphorylated and SrcKD-phosphorylated samples were analyzed alone and in the presence of CrkL, n = 3 to 10 independent experiments as indicated. Representative dissociation reactions are shown in (B) (WT) and in fig. S3 (A to D) (Cdc25HD and mutants). (D) Analysis by PD of the competition of CrkL with the Cdc25HD for binding to four constructs of the AIR that contain the P3 and P4 (GST-537- 646-WT), only the P3 (GST-537-646-P4A), only the P4 (GST-545-646-WT), or none of these proline-rich motifs (GST-545-646-P4A). PDs are representative of two independent experiments. (E) Time course of the in vitro phosphorylation of the AIR (537-646) by the SrcKD, analyzed by Western blot; representative of two independent experiments. (F) Binding of Cdc25HD to GST-AIR (537-646) phosphorylated with SrcKD in PD assays. PD is representative of two independent experiments. (G) Exchange activity (kobs) of the Cdc25HD alone and in the presence of AIR (untagged or GST-fusion) or the AIR mutants in (D). n = 3 to 4 independent experiments as indicated. Representative dissociation experiments are shown in fig. S3E. (H) Schematic representation of the effect of phosphorylation and CrkL binding to the AIR on the release of the inhibitory interaction when the AIR and the Cdc25HD are assayed as individual proteins.

Article Snippet: 13, eabb7075 (2020) 1 September 2020 13 of 17 rabbit pAb 121 against Rap1 (sc-65, Santa Cruz Biotechnology), and mouse mAb against -actin (dilution 1/2000; AC-15, SigmaAldrich).

Techniques: Activation Assay, Phospho-proteomics, Purification, Western Blot, Isolation, Binding Assay, Construct, In Vitro, Activity Assay

Journal: Science signaling

Article Title: Mechanisms of autoregulation of C3G, activator of the GTPase Rap1, and its catalytic deregulation in lymphomas.

doi: 10.1126/scisignal.abb7075

Figure Lengend Snippet: Fig. 7. The NTD/REM interaction stimulates the GEF activity of the Cdc25HD. (A) Schematic representation of full-length C3G-WT, truncation fragments, and the mutant E731R/E784R (red crosses) in which the NTD/REM interaction is destabilized. (B to E) Nucleotide exchange activity (kobs) of the proteins depicted in (A), in the absence of stimuli (B), in the presence of CrkL (C), phosphorylated with SrcKD (D), and when CrkL and phosphorylation were combined (E). The number of independent experiments (n) is indicated. Lines are means ± SD. Representative nucleotide dissociation reactions are shown in fig. S4 (A, B, D, and E). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by one-way ANOVA followed by Dunnett’s test (B and C) or unequal variance Brown-Forsythe ANOVA followed by Dunnett’s test (D and E). (F) Analysis of Rap1 activation in HEK293T cells expressing C3G-mEGFP WT, the mutant E731R/E784R, or C3G with the membrane targeting CAAX-tag. Results of two independent experiments are shown. (G) Schematic illustration of the contribution of the NTD/REM interaction to the activation by CrkL and phosphorylation of C3G WT and mutants.

Article Snippet: 13, eabb7075 (2020) 1 September 2020 13 of 17 rabbit pAb 121 against Rap1 (sc-65, Santa Cruz Biotechnology), and mouse mAb against -actin (dilution 1/2000; AC-15, SigmaAldrich).

Techniques: Activity Assay, Mutagenesis, Phospho-proteomics, Activation Assay, Expressing, Membrane