addgene egfp rac1 t17n  (Addgene inc)

Journal: bioRxiv

Article Title: Macropinocytosis of amyloid precursor protein requires the adaptor protein Fe65 and the recruitment and activity of Arf6 and the RhoGTPases Rac1, Cdc42 and RhoA

doi: 10.1101/2025.03.02.641070

Figure Lengend Snippet: A) N2a cells transfected with Rac1-EGFP (green), PLC8PH-mRFP (magenta) and APP695. Cells were incubated with tagged anti-APP or anti-NCAM antibodies (red) on ice and then were either immediately fixed on ice as a baseline (00:00), or incubated for 30 seconds (00:30), 2 minutes (02:00), 5 minutes (5:00), and 10 minutes (10:00) at 37°C 5% CO2 prior to fixation. Colocalization between signals is indicated by white pixels. B/C) Quantification of colocalization data (n=3; 15 images per replicate) is represented as the difference between the colocalization at each timepoint to the colocalization at baseline (mean % colocalized at XX:XX – mean % colocalized at 00:00). * denotes significant difference to baseline within condition; # denotes significant difference between groups at the indicated timepoint. D) N2a cells transfected with Cdc42-EGFP (green), PLC8PH-mRFP (magenta) and APP695. Experiment from A) was repeated with Cdc42-EGFP expressing cells. Colocalization between signals is indicated by white pixels. E/F) Quantification of colocalization data (n=3; 15 images per replicate). Data representation and analysis from B/C) was repeated for N2a cells expressing Cdc42-EGFP. * denotes significant difference to baseline within condition; # denotes significant difference between groups at the indicated timepoint. G) N2a cells transfected with RhoA-EGFP (green), PLC8PH-mRFP (magenta) and APP695. Experiment from A) was repeated with RhoA-EGFP expressing cells. Colocalization between signals is indicated by white pixels. H/I) Quantification of colocalization data (n=3; 15 images per replicate). Data representation and analysis from B/C) was repeated for N2a cells expressing RhoA-EGFP. * denotes significant difference to baseline within condition; # denotes significant difference between groups at the indicated timepoint. Significance was measured by a two-way ANOVA with a Tukey’s test using a single pooled variance. Data is presented as mean ± SEM. */# p<0.05; Scale bar = 5μm .

Article Snippet: Rac1-EGFP (pcDNA3.1-EGFP-Rac1(wt)) was a gift from Klaus Hahn (Addgene plasmid #13719).

Techniques: Transfection, Incubation, Expressing

Journal: bioRxiv

Article Title: Macropinocytosis of amyloid precursor protein requires the adaptor protein Fe65 and the recruitment and activity of Arf6 and the RhoGTPases Rac1, Cdc42 and RhoA

doi: 10.1101/2025.03.02.641070

Figure Lengend Snippet: A) G-LISA assay for Rac1-GTP from N2a cells incubated with no treatment, anti-NCAM antibodies, anti-APP antibodies or EGF (positive control) while on ice, then incubated for 5 minutes to allow for the initiation of macropinocytosis. Lysates were immediately collected and used for Rac1 G-LISA activation assays. To compare the relative amount of active GTP-bound Rac1 in each condition, the absolute optical density (OD) was measured from each condition’s lysate loaded into G-LISA assay wells. Three different lysate samples were collected for each condition from different biological samples (n=3), and the G-LISA assay was performed in technical triplicates. Significant differences from untreated samples were calculated by a one-way ANOVA with Tukey’s test. This same experiment and analysis were performed for B) Cdc42-GTP levels, and C) RhoA-GTP levels. Data is presented as mean ± SEM. *p<0.05 .

Article Snippet: Rac1-EGFP (pcDNA3.1-EGFP-Rac1(wt)) was a gift from Klaus Hahn (Addgene plasmid #13719).

Techniques: Incubation, Positive Control, Activation Assay

Journal: bioRxiv

Article Title: Macropinocytosis of amyloid precursor protein requires the adaptor protein Fe65 and the recruitment and activity of Arf6 and the RhoGTPases Rac1, Cdc42 and RhoA

doi: 10.1101/2025.03.02.641070

Figure Lengend Snippet: A) N2a cells transfected with either WT-APP (top) or APP-AENATA (bottom) and LAMP1-mCh (red). Cells were incubated with tagged anti-APP antibody (green) on ice for 20 minutes, then immediately fixed (0 min) or incubated for 15 minutes. Colocalization was assessed between bound/crosslinked APP and LAMP1 (white pixels). B) Quantification of the mean % of APP colocalized with LAMP1 from three replicate experiments (n=3; 15 images per replicate) of cells expressing WT APP or APP-AENATA. Statistical significance was analyzed by a one-way ANOVA with Tukey’s test. C) N2a cells transfected with WT-APP or APP-AENATA, Fe65-EGFP (green), and PLC8PH-mRFP (magenta). APP was bound/crosslinked by incubation with a tagged APP antibody (red) while on ice, then incubated for 30 seconds. Colocalization was measured between Fe65 and bound/crosslinked WT-APP or APP-AENATA, as well as between Fe65 and PLC8PH (white pixels). D) Quantification of the mean % of Fe65 colocalized with either bound/crosslinked APP (top) or PLC8PH (bottom) in N2a cells expressing WT-APP or APP-AENATA. Data presented comes from three replicate experiments (n=3; 15 images per replicate) of cells expressing WT APP or APP-AENATA. Significant differences between cells expressing WT or AENATA mutant APP was analyzed by a two-tailed unpaired t-test. This experiment was repeated with N2a cells expressing E/F) Arf6-GFP, G/H) Rac1-GFP, I/J) Cdc42-GFP, and K/L) RhoA-GFP. Data is presented as mean ± SEM. * p<0.05; Scale bar = 5 μm .

Article Snippet: Rac1-EGFP (pcDNA3.1-EGFP-Rac1(wt)) was a gift from Klaus Hahn (Addgene plasmid #13719).

Techniques: Transfection, Incubation, Expressing, Mutagenesis, Two Tailed Test

Journal: bioRxiv

Article Title: Macropinocytosis of amyloid precursor protein requires the adaptor protein Fe65 and the recruitment and activity of Arf6 and the RhoGTPases Rac1, Cdc42 and RhoA

doi: 10.1101/2025.03.02.641070

Figure Lengend Snippet: A) N2a cells transfected with Rac1-EGFP (green), PLC8PH-mRFP (magenta) and APP695. Cells were treated with 0.1% DMSO, 5μM NAV-2729, 10μM EHT 1864, 10μM ML 141, and 35μM Rhosin. After treatment, cells were incubated with tagged anti-APP antibodies (red) on ice and then fixed following a 30 second incubation. Colocalization was assessed between Rac1 and bound/crosslinked APP or PLC8PH (white pixels). B) Quantification of the mean % of Rac1 colocalized with APP (left) or PLC8PH (right) from three replicate experiments (n=3; 15 images per replicate). C) N2a cells transfected with Cdc42-EGFP (green), PLC8PH-mRFP (magenta) and APP695. Cells were treated as described in A), then incubated with tagged anti-APP antibodies (red) on ice and fixed following a 30 second incubation. Colocalization was assessed between Cdc42 and bound/crosslinked APP or PLC8PH (white pixels). D) Quantification of the mean % of Cdc42 colocalized with APP (left) or PLC8PH (right) from three replicate experiments (n=3; 15 images per replicate). E) N2a cells transfected with RhoA-EGFP (green), PLC8PH-mRFP (magenta) and APP695. Cells were treated as described in A), then incubated with tagged anti-APP antibodies (red) on ice and then fixed following an incubation for 30 seconds. Colocalization was assessed between RhoA and bound/crosslinked APP or PLC8PH (white pixels). F) Quantification of the mean % of RhoA colocalized with APP (left) or PLC8PH (right) from three replicate experiments (n=3; 15 images per replicate). Significant changes in the percentage colocalized between treatments were assessed by a one-way ANOVA with Tukey’s test . Data is presented as mean ± SEM. *p<0.05; Scale bar = 5μm .

Article Snippet: Rac1-EGFP (pcDNA3.1-EGFP-Rac1(wt)) was a gift from Klaus Hahn (Addgene plasmid #13719).

Techniques: Transfection, Incubation

Journal: bioRxiv

Article Title: Macropinocytosis of amyloid precursor protein requires the adaptor protein Fe65 and the recruitment and activity of Arf6 and the RhoGTPases Rac1, Cdc42 and RhoA

doi: 10.1101/2025.03.02.641070

Figure Lengend Snippet: A) G-LISA assay for Rac1-GTP from N2a cells treated with 0.1% DMSO, 5μM NAV-2729, 10μM EHT 1864, 10μM ML 141, and 35μM Rhosin as used in the experiments above. After treatment, lysates were immediately collected and used for Rac1 G-LISA activation assays. To compare the relative amount of active GTP-bound Rac1 in each treatment condition, the absolute optical density (OD) was measured from each condition’s lysate loaded into G-LISA assay wells. Three different lysate samples were collected for each condition from different biological samples (n=3), and the G-LISA assay was performed in technical triplicates. Significant differences from untreated samples were calculated by a one-way ANOVA with Tukey’s test. This same experiment and analysis was performed for B) Cdc42-GTP levels, and C) RhoA-GTP levels. Data is presented as mean ± SEM. *p<0.05 .

Article Snippet: Rac1-EGFP (pcDNA3.1-EGFP-Rac1(wt)) was a gift from Klaus Hahn (Addgene plasmid #13719).

Techniques: Activation Assay

Journal: Life Science Alliance

Article Title: Noncanonical altPIDD1 protein: unveiling the true major translational output of the PIDD1 gene

doi: 10.26508/lsa.202402910

Figure Lengend Snippet: (A) Images by confocal microscopy of altPIDD1 (HA tag, green) and PIDD1 (Flag tag, red) in HeLa cells transfected with PIDD1 (HA)Flag . Identically stained mock-transfected cells did not display any signal, highlighting the specificity of the observed signals. The white scale bar corresponds to 10 μm. Representative images of n = 3. Arrows indicate accumulation of altPIDD1 in cytoplasmic and membrane filamentous structures. (B) Images by confocal microscopy of altPIDD1 (HA tag, green) and PIDD1 (flag tag, red) in HeLa cells transfected with altPIDD1 HA and PIDD1 FLAG , respectively. The white scale bar corresponds to 10 μm. Representative images of n = 3. Arrows indicate accumulation of altPIDD1 in cytoplasmic and membrane structures. (C) Images by confocal microscopy of altPIDD1 (HA tag, green) in cells labeled with phalloidin to show actin structures in migrating HeLa cells. Migration was induced by scratching a confluent cell layer 24 h before fixation. Left panel, cell with high levels of stress fibers (arrows). Right panel, cell with a large lamellipodium (arrows). The white scale bar corresponds to 10 μm. Representative images of n = 3. (D) Images by confocal microscopy of altPIDD1 (HA tag, green) and actin (phalloidin, red) in HeLa cells co-transfected with either RhoA-Q63L or Rac1-Q61L cells, as indicated, to induce the formation of stress fibers and lamellipodia, respectively. The white scale bar corresponds to 10 μm. Representative images of n = 3.

Article Snippet: All constructs were confirmed by sequencing. pcDNA3-EGFP-RhoA-Q63L was a gift from Gary Bokoch (plasmid # 12968; http://n2t.net/addgene:12968 ; RRID:Addgene_12968; Addgene). pcDNA3-EGFP-Rac1-Q61L was a gift from Gary Bokoch (plasmid # 12981; http://n2t.net/addgene:12981 ; RRID:Addgene_12981; Addgene).

Techniques: Confocal Microscopy, FLAG-tag, Transfection, Staining, Membrane, Labeling, Migration

Journal: Cell

Article Title: Targeting Ras-, Rho-, and Rab-family GTPases via a conserved cryptic pocket

doi: 10.1016/j.cell.2024.08.017

Figure Lengend Snippet:

Article Snippet: pcDNA3.1(+) EGFP-Rac1 (G12C) , This study , Addgene #224279.

Techniques: Virus, Recombinant, Protease Inhibitor, Staining, Activation Assay, BIA-KA, Mass Spectrometry, Plasmid Preparation, Software, Control, Transfection, Western Blot

Journal: Cell

Article Title: Targeting Ras-, Rho-, and Rab-family GTPases via a conserved cryptic pocket

doi: 10.1016/j.cell.2024.08.017

Figure Lengend Snippet:

Article Snippet: pcDNA3.1(+) EGFP-Rac1 (P29S) , This study , Addgene #224280.

Techniques: Virus, Recombinant, Protease Inhibitor, Staining, Activation Assay, BIA-KA, Mass Spectrometry, Plasmid Preparation, Software, Control, Transfection, Western Blot

Journal: Cell

Article Title: Targeting Ras-, Rho-, and Rab-family GTPases via a conserved cryptic pocket

doi: 10.1016/j.cell.2024.08.017

Figure Lengend Snippet:

Article Snippet: pcDNA3.1(+) EGFP-Rac1 (WT) , This study , Addgene #224278.

Techniques: Virus, Recombinant, Protease Inhibitor, Staining, Activation Assay, BIA-KA, Mass Spectrometry, Plasmid Preparation, Software, Control, Transfection, Western Blot