milk ppak1 2 t423 402 ab 330220 cell signaling technology  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: p21-activated kinase regulates Rab3a vesicles to repair plasma membrane damage caused by Amyloid-β oligomers

doi: 10.1101/2025.02.11.637764

Figure Lengend Snippet: TEM images of A) oAβ 1-40 and B) oAβ 1-42 . C, D) Fluorescently (TMR, red) labelled C) oAβ 1-40 and D) oAβ 1-42 end up to lysotracker positive vesicles (green). E) Percentage of colocalization of TMR-labelled oAβ peptides with lysotracker positive vesicles after 2 hours of exposure, and F) Size of oAβ accumulated lysotracker positive endolysosomes. G) Internalization of oAβ 1-40 -TMR and oAβ 1- 42 -TMR into the EEA1 positive early endosomal vesicles. H) Graphical representation of oAβ peptides colocalization with EEA1. I) WBs of EEA1 Aβ 1-42 treated cells with and without IPA-3. J) Quantification of EEA1 expression. K,L) Lamp1in K) Aβ 1-40 and L) Aβ 1-42 treated cells, with and without IPA-3, a specific inhibitor of pPAK1. M) Quantification of Lamp1 levels. Data are expressed as mean ± SD, *** p ≤ 0.001. Statistical significance was validated using the student-t test for E and F, and one-way ANOVA for I and K. n=3.

Article Snippet: Differential immunostainings were performed to gain insights into actin modulation following membrane damage and the distribution of Rab3a for PM repair ( , ), using pPAK1[phospho-PAK1] (Thr423)/PAK2 (Thr402) antibody (Cell Signaling Technology; CST #2601); f-actin binding phallotoxin phalloidin (A30106, Invitrogen); EEA1 [E4156, Sigmaaldrich]; Lamp1 (PE Conjugated, 2293862, Invitrogen) and Rab3a (PA1-770, Invitrogen).

Techniques: Expressing

Journal: bioRxiv

Article Title: p21-activated kinase regulates Rab3a vesicles to repair plasma membrane damage caused by Amyloid-β oligomers

doi: 10.1101/2025.02.11.637764

Figure Lengend Snippet: WBs of A, B) pPAK1 for Aβ 1-40 and Aβ 1-42 treated cells with and without IPA3 treatment. Quantification of C) pPAK1 levels. WBs of D, E) Rab3a for Aβ 1-40 and Aβ 1-42 treated cells with and without IPA3 treatment. Quantification of F) Rab3a levels. The data are presented as mean ± SD, with *** denoting significance at p ≤ 0.001. The statistical analysis was performed using one-way ANOVA.

Article Snippet: Differential immunostainings were performed to gain insights into actin modulation following membrane damage and the distribution of Rab3a for PM repair ( , ), using pPAK1[phospho-PAK1] (Thr423)/PAK2 (Thr402) antibody (Cell Signaling Technology; CST #2601); f-actin binding phallotoxin phalloidin (A30106, Invitrogen); EEA1 [E4156, Sigmaaldrich]; Lamp1 (PE Conjugated, 2293862, Invitrogen) and Rab3a (PA1-770, Invitrogen).

Techniques:

Journal: bioRxiv

Article Title: p21-activated kinase regulates Rab3a vesicles to repair plasma membrane damage caused by Amyloid-β oligomers

doi: 10.1101/2025.02.11.637764

Figure Lengend Snippet: A) Knockdown efficiency was assessed through the WB experiment conducted on pLKO.1 lentiviral vector-transfected cells as a control and cells with Rab3a knockdown (shRab3A). B) WB of total-PAK1 and pPAK1 on pLKO.1 lentiviral vector-transfected cells as a control and cells with Rab3a knockdown (shRab3A). The quantifications are presented as mean ± SD, with *** indicating p ≤ 0.001. Statistical analysis employed a t-test, and n=3. C-D) Dual nuclear staining using Propidium iodide and Hoechst was performed on C) pLKO.1 and D) shRab3a transfected cells, treated with Aβ 1-42 for 30 minutes and 1 hour. E) Graphical representation of PI-positive cells per unit area percentage in both pLKO.1 and shRab3a transfected cells. F) Cell viability by MTT assay was performed in pLKO.1 and shRab3a transfected cells, treated with and without Aβ 1-42 for 3 and 6 hours. The data is expressed as mean ± SD, with *** indicating p ≤ 0.001. One-way ANOVA was used for statistical analysis. Statistical analysis involved ordinary two-way ANOVA, and the sample size was 9 from three independent experiments (n=3).

Article Snippet: Differential immunostainings were performed to gain insights into actin modulation following membrane damage and the distribution of Rab3a for PM repair ( , ), using pPAK1[phospho-PAK1] (Thr423)/PAK2 (Thr402) antibody (Cell Signaling Technology; CST #2601); f-actin binding phallotoxin phalloidin (A30106, Invitrogen); EEA1 [E4156, Sigmaaldrich]; Lamp1 (PE Conjugated, 2293862, Invitrogen) and Rab3a (PA1-770, Invitrogen).

Techniques: Knockdown, Plasmid Preparation, Transfection, Control, Staining, MTT Assay

Journal: bioRxiv

Article Title: p21-activated kinase regulates Rab3a vesicles to repair plasma membrane damage caused by Amyloid-β oligomers

doi: 10.1101/2025.02.11.637764

Figure Lengend Snippet: The extracellular oAβ induces PM damage: oAβ 1-40 causes less damage, while oAβ 1-42 causes higher damage. The PM repair occurs via the Rab3-dependent recycling of Lamp1-positive lysosomal vesicles coupled with pPAK1-mediated massive endocytosis of the oAβ peptides. PM repair, in response to oAβ1-42 induced damage, occurs at a much faster rate. pPAK1 regulates actin-membrane stress via controlling CIE and by forming long-stretched fibrillary actin conduits or TNT-like structures during rapid PM repair.

Article Snippet: Differential immunostainings were performed to gain insights into actin modulation following membrane damage and the distribution of Rab3a for PM repair ( , ), using pPAK1[phospho-PAK1] (Thr423)/PAK2 (Thr402) antibody (Cell Signaling Technology; CST #2601); f-actin binding phallotoxin phalloidin (A30106, Invitrogen); EEA1 [E4156, Sigmaaldrich]; Lamp1 (PE Conjugated, 2293862, Invitrogen) and Rab3a (PA1-770, Invitrogen).

Techniques: Membrane

Journal: bioRxiv

Article Title: p21-activated kinase regulates Rab3a vesicles to repair plasma membrane damage caused by Amyloid-β oligomers

doi: 10.1101/2025.02.11.637764

Figure Lengend Snippet: TEM images of A) oAβ 1-40 and B) oAβ 1-42 . C, D) Fluorescently (TMR, red) labelled C) oAβ 1-40 and D) oAβ 1-42 end up to lysotracker positive vesicles (green). E) Percentage of colocalization of TMR-labelled oAβ peptides with lysotracker positive vesicles after 2 hours of exposure, and F) Size of oAβ accumulated lysotracker positive endolysosomes. G) Internalization of oAβ 1-40 -TMR and oAβ 1- 42 -TMR into the EEA1 positive early endosomal vesicles. H) Graphical representation of oAβ peptides colocalization with EEA1. I) WBs of EEA1 Aβ 1-42 treated cells with and without IPA-3. J) Quantification of EEA1 expression. K,L) Lamp1in K) Aβ 1-40 and L) Aβ 1-42 treated cells, with and without IPA-3, a specific inhibitor of pPAK1. M) Quantification of Lamp1 levels. Data are expressed as mean ± SD, *** p ≤ 0.001. Statistical significance was validated using the student-t test for E and F, and one-way ANOVA for I and K. n=3.

Article Snippet: Next, the membrane underwent overnight incubation at 4°C with constant agitation, utilizing antibodies against pPAK1 [Cell Signaling Technology, #2601, (1:750)], Rab3a [PA1-770, Invitrogen (1:750)], Lamp1 [611042, BD transduction Lab, (1:750)], and EEA1 [E4156, Sigmaaldrich] (1;750), with loading control antibodies directed at actin (PAS097Hu01, Cloud Clone) or GAPDH (CAB932Hu22, Cloud Clone) in 1:1000.

Techniques: Expressing

Journal: bioRxiv

Article Title: p21-activated kinase regulates Rab3a vesicles to repair plasma membrane damage caused by Amyloid-β oligomers

doi: 10.1101/2025.02.11.637764

Figure Lengend Snippet: WBs of A, B) pPAK1 for Aβ 1-40 and Aβ 1-42 treated cells with and without IPA3 treatment. Quantification of C) pPAK1 levels. WBs of D, E) Rab3a for Aβ 1-40 and Aβ 1-42 treated cells with and without IPA3 treatment. Quantification of F) Rab3a levels. The data are presented as mean ± SD, with *** denoting significance at p ≤ 0.001. The statistical analysis was performed using one-way ANOVA.

Article Snippet: Next, the membrane underwent overnight incubation at 4°C with constant agitation, utilizing antibodies against pPAK1 [Cell Signaling Technology, #2601, (1:750)], Rab3a [PA1-770, Invitrogen (1:750)], Lamp1 [611042, BD transduction Lab, (1:750)], and EEA1 [E4156, Sigmaaldrich] (1;750), with loading control antibodies directed at actin (PAS097Hu01, Cloud Clone) or GAPDH (CAB932Hu22, Cloud Clone) in 1:1000.

Techniques:

Journal: bioRxiv

Article Title: p21-activated kinase regulates Rab3a vesicles to repair plasma membrane damage caused by Amyloid-β oligomers

doi: 10.1101/2025.02.11.637764

Figure Lengend Snippet: A) Knockdown efficiency was assessed through the WB experiment conducted on pLKO.1 lentiviral vector-transfected cells as a control and cells with Rab3a knockdown (shRab3A). B) WB of total-PAK1 and pPAK1 on pLKO.1 lentiviral vector-transfected cells as a control and cells with Rab3a knockdown (shRab3A). The quantifications are presented as mean ± SD, with *** indicating p ≤ 0.001. Statistical analysis employed a t-test, and n=3. C-D) Dual nuclear staining using Propidium iodide and Hoechst was performed on C) pLKO.1 and D) shRab3a transfected cells, treated with Aβ 1-42 for 30 minutes and 1 hour. E) Graphical representation of PI-positive cells per unit area percentage in both pLKO.1 and shRab3a transfected cells. F) Cell viability by MTT assay was performed in pLKO.1 and shRab3a transfected cells, treated with and without Aβ 1-42 for 3 and 6 hours. The data is expressed as mean ± SD, with *** indicating p ≤ 0.001. One-way ANOVA was used for statistical analysis. Statistical analysis involved ordinary two-way ANOVA, and the sample size was 9 from three independent experiments (n=3).

Article Snippet: Next, the membrane underwent overnight incubation at 4°C with constant agitation, utilizing antibodies against pPAK1 [Cell Signaling Technology, #2601, (1:750)], Rab3a [PA1-770, Invitrogen (1:750)], Lamp1 [611042, BD transduction Lab, (1:750)], and EEA1 [E4156, Sigmaaldrich] (1;750), with loading control antibodies directed at actin (PAS097Hu01, Cloud Clone) or GAPDH (CAB932Hu22, Cloud Clone) in 1:1000.

Techniques: Knockdown, Plasmid Preparation, Transfection, Control, Staining, MTT Assay

Journal: bioRxiv

Article Title: p21-activated kinase regulates Rab3a vesicles to repair plasma membrane damage caused by Amyloid-β oligomers

doi: 10.1101/2025.02.11.637764

Figure Lengend Snippet: The extracellular oAβ induces PM damage: oAβ 1-40 causes less damage, while oAβ 1-42 causes higher damage. The PM repair occurs via the Rab3-dependent recycling of Lamp1-positive lysosomal vesicles coupled with pPAK1-mediated massive endocytosis of the oAβ peptides. PM repair, in response to oAβ1-42 induced damage, occurs at a much faster rate. pPAK1 regulates actin-membrane stress via controlling CIE and by forming long-stretched fibrillary actin conduits or TNT-like structures during rapid PM repair.

Article Snippet: Next, the membrane underwent overnight incubation at 4°C with constant agitation, utilizing antibodies against pPAK1 [Cell Signaling Technology, #2601, (1:750)], Rab3a [PA1-770, Invitrogen (1:750)], Lamp1 [611042, BD transduction Lab, (1:750)], and EEA1 [E4156, Sigmaaldrich] (1;750), with loading control antibodies directed at actin (PAS097Hu01, Cloud Clone) or GAPDH (CAB932Hu22, Cloud Clone) in 1:1000.

Techniques: Membrane

Journal: bioRxiv

Article Title: The phosphorylation of Pak1 by Erk1/2 for cell migration requires Arl4D acting as a scaffolding protein

doi: 10.1101/2024.12.02.626348

Figure Lengend Snippet: (A) NIH3T3 and A10 cells, after knocked down with siCtrl, mouse siArl4A, or mouse siArl4D RNA, were serum starved and treated with PDGF-BB (20 ng/mL) for 10 min. Cell lysates immediately harvested were subjected to SDS-PAGE and Western blotting for indicated proteins. The red arrow indicates the position of Arl4D. The pPak1 T212 signals were divided with total Pak1 signals and normalized with the value of the first control group. The quantified results are shown in the dot plots with error bars indicating the mean ± SD (NIH/3T3, n=4; A-10, n=3). *P<0.05; ***P<0.001; ****P<0.0001 (one-way ANOVA with Tukey’s post hoc multiple comparison test). (B) NIH3T3 cells knocked down with siCtrl or mouse siArl4D RNA and transfected with empty vector pSG5 or human Arl4D-WT or Arl4D-G2A were serum starved and treated with PDGF-BB (20 ng/mL) for 10 min. Cell lysates immediately harvested were subjected to Western blotting for indicated proteins. The pPak1 T212 signals were divided with the total Pak1 signals and normalized with the value of the first control group. The quantified results are shown in the dot plots with the error bars indicating the mean ± SD (n=3). **P<0.01; ***P<0.001 (one-way ANOVA with Tukey’s post hoc multiple comparison test). (C) NIH3T3 cells transfected with empty vector pSG5, Arl4D-WT-HA or Arl4D-G2A-HA were serum starved and treated with PDGF-BB at 20 ng/mL for 10 min. Immediately harvested cell lysates were subjected to Western blotting for indicated proteins. The pPak1 T212 signals were divided with total Pak1 signals and normalized with the value of the first control group. The quantified results are shown in the dot plots with the error bars indicating the mean ± SD (n=4). **P<0.01; ***P<0.001; ****P< 0.0001 (one-way ANOVA with Tukey’s post hoc multiple comparison test).

Article Snippet: For Western blotting, the following primary antibodies were used at the indicated dilutions: anti-α-tubulin (1:10000, Sigma), anti-His (1:5000, #631212, Takara), anti-Erk1/2 (1:2000, #4695S, Cell Signaling), anti-pErk1/2 (1:2000, #4370S, Cell Signaling), anti-Pak1 (1:2000, #2602S, Cell Signaling), anti-pPak1 T212 (1:1000, #PA5-37677, Invitrogen), anti-Myc (1:3000, #2278S, Cell Signaling), anti-HA (1:3000, #3724S, Cell Signaling), anti-Na + /K + ATPase (1:1000, #3010S, Cell Signaling), anti-Lamin A/C (1:2000, #4777S, Cell Signaling), anti-Arl4D (1:1000, boosted from our lab), anti-GFP (1:1000, boosted from our lab), anti-Calnexin (1:1000, boosted from our lab) and anti-ϕ3-actin (1:2000, #GTX629630, GeneTex).

Techniques: SDS Page, Western Blot, Control, Comparison, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: The phosphorylation of Pak1 by Erk1/2 for cell migration requires Arl4D acting as a scaffolding protein

doi: 10.1101/2024.12.02.626348

Figure Lengend Snippet: Under PDGF treatment, Arl4D plays the important role in interacting and recruiting activated Erk1/2 as well as Pak1 to the plasma membrane. Following, Arl4D serves as the interbridge between activated Erk1/2 and Pak1 to make Pak1 T212 motif be phosphorylated successfully, which promote the cell motility.

Article Snippet: For Western blotting, the following primary antibodies were used at the indicated dilutions: anti-α-tubulin (1:10000, Sigma), anti-His (1:5000, #631212, Takara), anti-Erk1/2 (1:2000, #4695S, Cell Signaling), anti-pErk1/2 (1:2000, #4370S, Cell Signaling), anti-Pak1 (1:2000, #2602S, Cell Signaling), anti-pPak1 T212 (1:1000, #PA5-37677, Invitrogen), anti-Myc (1:3000, #2278S, Cell Signaling), anti-HA (1:3000, #3724S, Cell Signaling), anti-Na + /K + ATPase (1:1000, #3010S, Cell Signaling), anti-Lamin A/C (1:2000, #4777S, Cell Signaling), anti-Arl4D (1:1000, boosted from our lab), anti-GFP (1:1000, boosted from our lab), anti-Calnexin (1:1000, boosted from our lab) and anti-ϕ3-actin (1:2000, #GTX629630, GeneTex).

Techniques: Membrane