Journal: Cell reports

Article Title: FARP1, ARHGEF39, and TIAM2 are essential receptor tyrosine kinase effectors for Rac1-dependent cell motility in human lung adenocarcinoma

doi: 10.1016/j.celrep.2021.109905

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: These accession numbers for the datasets are listed in the . table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-AKT S473 Cell Signaling Technology Cat# 4060; RRID:AB_2315049 Anti-AKT Cell Signaling Technology Cat# 4691; RRID:AB_915783 Anti-phospho-PAK Cell Signaling Technology Cat# 2605; RRID:AB_2160222 Anti-PAK1/2/3 Cell Signaling Technology Cat# 2604;RRID:AB_2160225 Anti-AXL Cell Signaling Technology Cat# 8661; RRID:AB_11217435 Anti-AXL Y702 Cell Signaling Technology Cat# 5724; RRID:AB_10544794 Anti-Gab1 Cell Signaling Technology Cat# 3232; RRID:AB_2304999 Anti-Gab1 Y659 Cell Signaling Technology Cat# 12745; RRID:AB_2798014 Anti-Grb2 Cell Signaling Technology Cat# 3972; RRID:AB_10693935 Anti-SHP2 Cell Signaling Technology Cat# 3752; RRID:AB_2300607 Anti-Rac1clone 23A8 Millipore Sigma Cat# 05-389; RRID:AB_309712 Anti-RhoG Cell Signaling Technology 60370 Anti-β-actin Millipore Sigma Cat# A5441; RRID:AB_476744 Anti-cortactin Santa Cruz Cat# sc-11408; RRID:AB_2088281 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 Invitrogen Cat# {"type":"entrez-nucleotide","attrs":{"text":"R37117","term_id":"794573","term_text":"R37117"}} R37117 , RRID:AB_2556545 Anti-rabbit IgG, HRP-linked Bio-Rad Cat# 170-6515, RRID:AB_11125142 Anti-mouse IgG HRP-linked Bio-Rad Cat# 170-6516, RRID:AB_11125547 Biological samples Tumor samples from patients with lung adenocarcinoma University of Pennsylvania (IRB protocol # 805800).

Techniques: Recombinant, Staining, Software, Sequencing

Journal: Cell reports

Article Title: FARP1, ARHGEF39, and TIAM2 are essential receptor tyrosine kinase effectors for Rac1-dependent cell motility in human lung adenocarcinoma

doi: 10.1016/j.celrep.2021.109905

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: These accession numbers for the datasets are listed in the . table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-AKT S473 Cell Signaling Technology Cat# 4060; RRID:AB_2315049 Anti-AKT Cell Signaling Technology Cat# 4691; RRID:AB_915783 Anti-phospho-PAK Cell Signaling Technology Cat# 2605; RRID:AB_2160222 Anti-PAK1/2/3 Cell Signaling Technology Cat# 2604;RRID:AB_2160225 Anti-AXL Cell Signaling Technology Cat# 8661; RRID:AB_11217435 Anti-AXL Y702 Cell Signaling Technology Cat# 5724; RRID:AB_10544794 Anti-Gab1 Cell Signaling Technology Cat# 3232; RRID:AB_2304999 Anti-Gab1 Y659 Cell Signaling Technology Cat# 12745; RRID:AB_2798014 Anti-Grb2 Cell Signaling Technology Cat# 3972; RRID:AB_10693935 Anti-SHP2 Cell Signaling Technology Cat# 3752; RRID:AB_2300607 Anti-Rac1clone 23A8 Millipore Sigma Cat# 05-389; RRID:AB_309712 Anti-RhoG Cell Signaling Technology 60370 Anti-β-actin Millipore Sigma Cat# A5441; RRID:AB_476744 Anti-cortactin Santa Cruz Cat# sc-11408; RRID:AB_2088281 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 Invitrogen Cat# {"type":"entrez-nucleotide","attrs":{"text":"R37117","term_id":"794573","term_text":"R37117"}} R37117 , RRID:AB_2556545 Anti-rabbit IgG, HRP-linked Bio-Rad Cat# 170-6515, RRID:AB_11125142 Anti-mouse IgG HRP-linked Bio-Rad Cat# 170-6516, RRID:AB_11125547 Biological samples Tumor samples from patients with lung adenocarcinoma University of Pennsylvania (IRB protocol # 805800).

Techniques: Recombinant, Staining, Software, Sequencing

Journal: iScience

Article Title: PAK1 contributes to cerebral ischemia/reperfusion injury by regulating the blood-brain barrier integrity

doi: 10.1016/j.isci.2023.107333

Figure Lengend Snippet: Focal cerebral ischemia increases PAK1 phosphorylation (A) Representative image of blood flow measured by laser speckle flowmeter. (B) The time-dependent changes of PAK1 and p-PAK1 in ischemic cortex at 1 h, 6 h, 12 h, or 24 h reperfusion after 1-h transient middle cerebral artery occlusion (tMCAO). The homogenates of cortical brains from tMCAO and sham treated mice were subjected to Western blot analysis using indicated antibodies. GAPDH was used as a loading control. (C and D) The quantitative analysis of immunoblotted p-PAK1 (Ser144) and PAK1 proteins. Data are expressed as mean ± SEM (n = 4). ∗p < 0.05 versus sham, one-way ANOVA with Dunnet’s post hoc test. (E) Immunofluorescence images showing the colocalization of PAK1-positive (green) with microvessel markers Lectin (red) in the ipsilateral (I)/contralateral (C) cortex at 1 h reperfusion after tMCAO. White arrow shows that PAK1 is localized on blood vessels. Nuclei were stained with DAPI (blue). Scale bar = 20 μm. See also Figure S1 .

Article Snippet: Rabbit polyclonal anti-p-PAK1 (Ser144) , Cell Signaling Technology , Cat#2606; RRID: AB_2299279.

Techniques: Phospho-proteomics, Western Blot, Control, Immunofluorescence, Staining

Journal: iScience

Article Title: PAK1 contributes to cerebral ischemia/reperfusion injury by regulating the blood-brain barrier integrity

doi: 10.1016/j.isci.2023.107333

Figure Lengend Snippet: PAK1 inhibition by its inhibitors or siRNA reduces the hyperpermeability of bEnd.3 endothelial monolayer in OGD model (A) Immunoblots of p-PAK1 and PAK1 with corresponding antibodies in bEnd.3 cells at reperfusion for indicated times after 2 h OGD. GAPDH was used as a loading control. (B and C) The statistical analysis of immunoblotted p-PAK1 (Ser144) and PAK1 proteins. Data are expressed as mean ± SEM (n = 4). ∗p < 0.05, ∗∗p < 0.01 versus control groups, one-way ANOVA with Dunnet’s post hoc test. (D) Schematic representation of the in vitro BBB model. bEnd.3 cells were planted on microporous membrane of transwell inserts till confluence. After OGD/R treatment, the fluorescence dyes were added to upper compartment and the fluorescence intensity of lower compartment was measured 30 min later. (E and F) The transfer rate of dextran from upper compartment to lower compartment was examined to assess the endothelial monolayer permeability. Consistent diffused 4.4 kDa TRITC-dextran or 70 kDa FITC-dextran during reoxygenation after 2 h OGD was limited by FRAX486 (1 μmol/L) or IPA-3 (10 μmol/L) treatment. Data are expressed as mean ± SEM (n = 5). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus ctrl group; #p < 0.05, ##p < 0.01, ###p < 0.001 versus OGD + vehicle group, two-way ANOVA with Turkey’s post hoc test. (G) The endothelial monolayer permeability was further expressed as coefficient of diffusion (in centimeters per second). FRAX486 (1 μmol/L) or IPA-3 (10 μmol/L) treatment significantly reduced the increased permeability of bEnd.3 monolayer to 4.4 kDa TRITC-dextran or 70 kDa FITC-dextran by exposure to OGD/R 3 h. Data are expressed as mean ± SEM (n = 5). ∗∗∗p < 0.001 versus ctrl group; #p < 0.05 versus OGD + vehicle (veh) group, one-way ANOVA with Sidak’s post hoc test. (H) PAK1 expression in bEnd.3 cells is downregulated by specific siPAK1, not siNC. Data are expressed as mean ± SEM (n = 3). ∗∗∗p < 0.001, Student’s t test. (I) Increased endothelial monolayer permeability of bEnd.3 cells to 4.4 kDa TRITC-dextran or 70 kDa FITC-dextran at OGD/R 3h was inhibited by siPAK1. Data are expressed as mean ± SEM (n = 5). ∗∗p < 0.01 versus ctrl group; #p < 0.05 versus OGD + siNC group, one-way ANOVA with Sidak’s post hoc test. See also Figure S2 .

Article Snippet: Rabbit polyclonal anti-p-PAK1 (Ser144) , Cell Signaling Technology , Cat#2606; RRID: AB_2299279.

Techniques: Inhibition, Western Blot, Control, In Vitro, Membrane, Fluorescence, Permeability, Diffusion-based Assay, Expressing

Journal: iScience

Article Title: PAK1 contributes to cerebral ischemia/reperfusion injury by regulating the blood-brain barrier integrity

doi: 10.1016/j.isci.2023.107333

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-p-PAK1 (Ser144) , Cell Signaling Technology , Cat#2606; RRID: AB_2299279.

Techniques: Recombinant, Plasmid Preparation, Lysis, Bicinchoninic Acid Protein Assay, Protease Inhibitor, Cell Counting, Negative Control, Software

Journal: Stem cells (Dayton, Ohio)

Article Title: Pak2 regulates hematopoietic progenitor cell proliferation, survival and differentiation

doi: 10.1002/stem.1951

Figure Lengend Snippet: (A) Schematic of competitive repopulation transplantation. (B) Pak2 expression in sorted CD45.2+ BM cells at 6 months post-polylC administrations. The percentage of peripheral blood CD45.2+ cells in lethally irradiated (C) primary and (D) secondary recipients. N=6-8 and n=9-11 mice/group in primary and secondary transplantation experiments, respectively. Representative data from 3 competitive transplantation experiments are shown. Student t test, * indicates p<0.05 between Pak2-KO and WT. In secondary transplantation experiment, 0 vs 6 weeks: p>0.05 in both WT and Pak2-KO cohorts.

Article Snippet: 23 Primary antibody was rabbit anti-Pak2 polyclonal antibody (Cell signaling Technology, Danvers, MA).

Techniques: Transplantation Assay, Expressing, Irradiation

Journal: Stem cells (Dayton, Ohio)

Article Title: Pak2 regulates hematopoietic progenitor cell proliferation, survival and differentiation

doi: 10.1002/stem.1951

Figure Lengend Snippet: CD45.1+ WT BoyJ mice were non-competitively transplanted with CD45.2+Pak2flox/floxMx1Cre+ or Pak2flox/floxMx1Cre− BM cells. Peripheral blood was collected at indicated time points post polylC treatment for complete blood cell count (CBC) and flow cytometry. (A) total white blood cell (WBC) count, (B) the absolute number of CD45.2+ WBCs in the peripheral blood (C) hemoglobin (Hgb) level, (D) mean corpuscular volume (MCV) of red blood cells. N=6-8/group. Representative data from 3 experiments are shown. Student t test, * indicates p<0.05 between Pak2-KO and WT.

Article Snippet: 23 Primary antibody was rabbit anti-Pak2 polyclonal antibody (Cell signaling Technology, Danvers, MA).

Techniques: Cell Counting, Flow Cytometry

Journal: Stem cells (Dayton, Ohio)

Article Title: Pak2 regulates hematopoietic progenitor cell proliferation, survival and differentiation

doi: 10.1002/stem.1951

Figure Lengend Snippet: Flow cytometric and colonogenic analysis of BM in mice that were competitively transplanted with the 1:1 mix of CD45.1+ BoyJ and CD45.2+Pak2flox/floxMx1Cre+ (Pak2-KO) or Pak2flox/floxMx1Cre− (WT) BM cells. (A) The percentage and (B) absolute number of CD45.2+ phenotypic HPCs (Lin−Sca1+c-Kit+) in the BM. (C) The absolute number of CD45.2+ multi-cytokine stimulated colonies which represent the sum of CFU-GM, CFU-GEMM and BFU-E per femur. (D) The percentage of CD45.2+ multi-cytokine stimulated colonies that were in S phase as determined by high specific activity tritiated thymidine suicide assays. (E) The percentage and (F) absolute number of phenotypic CD45.2+ HSCs (Lin−Sca1+c-Kit+CD150+CD48/41−) in the BM. Representative data from 2-4 independent experiments are shown, 3 or more mice per genotype in each experiment. Student t test. ns=non-significant, p>0.05. Data was collected at 6 to 8 months post PIPC administration.

Article Snippet: 23 Primary antibody was rabbit anti-Pak2 polyclonal antibody (Cell signaling Technology, Danvers, MA).

Techniques: Activity Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Pak2 regulates hematopoietic progenitor cell proliferation, survival and differentiation

doi: 10.1002/stem.1951

Figure Lengend Snippet: (A) The percentage of peripheral blood CD45.2+ myeloid cells (B220−CD3−), lymphocytes (combining CD3+ and B220+ gates), PMNs (B220−CD3−Mac-1highLy6Ghigh) and monocytes (B220−CD3−Mac-1+Ly6Glow) is illustrated in panel (i), (ii), (iii) and (iv), respectively. N=6-10 mice/group. Student t test, * indicates p<0.001 between Pak2-KO and WT. (B) The percentage of granulocyte-monocyte progenitors (GMP: Lin−Sca1−c-Kit+CD34+FcγRII/III+), megakaryocyte-erythroid progenitors (MEP: Lin−Sca1−c-Kit+CD34TcγRII/III−) and common myeloid progenitors (CMP: Lin−Sca1−c-Kit+CD34+FcγRII/III−/low) within the myeloid progenitor (MP: Lin−Sca1−c-Kit+) population. N=5-6 mice/group. Two way ANOVA, p<0.001. (C) Colony formation of sorted CD45.2+ BM LDMNCs in the presence of GM-CSF. N=6-10 mice/group. Student t test, p<0.05 between Pak2-KO and WT. (D) 106 lentiviral vector transduced Pak2flox/flox c-kit+ cells were cultured in murine GM-CSF for 7 days prior to Mac-1 and Ly6G staining. Absolute number of PMNs (Mac-1highLy6Ghigh) is shown. Student t test, p<0.05. (E) cytospin preparations of the progenies from the multi-cytokine stimulated colony assays shown in figure 4C. Arrows indicate PMNs. Panel (i) and (ii) illustrate representative images under 100× magnification; panel (iii) shows the percentage of PMNs. Total of 1093 and 837 progenies were counted from WT and Pak2-KO colonies, respectively. N=6. Student t test, p<0.05 (F) Peripheral blood smears from mice transplanted with Pak2-KO ad WT BM (magnification 100×). (i) Percentage of hypersegmented neutrophils (n=3 mice/group), (ii) A normal neutrophil with a typical doughnut-shaped nucleus, (iii) to (v) Representative Pak2-KO neutrophils with hypersegmented nucleus. Representative data from at least 2 experiments are shown.

Article Snippet: 23 Primary antibody was rabbit anti-Pak2 polyclonal antibody (Cell signaling Technology, Danvers, MA).

Techniques: Plasmid Preparation, Cell Culture, Staining

Journal: Stem cells (Dayton, Ohio)

Article Title: Pak2 regulates hematopoietic progenitor cell proliferation, survival and differentiation

doi: 10.1002/stem.1951

Figure Lengend Snippet: A) Absolute number of CD45.2+ common lymphoid progenitors (CLPs: Lin−IL7Ra+c-KitlowSca1low), (B) number of CD45.2+CD3+ T cells in thymus, (C) percentage of CD4+CD8+, CD4+CD8−, CD4−CD8+ and CD4−CD8− cells within the CD45.2+CD3+ thymic T cell population, (D) number of CD45.2+CD3+ T cells in spleen, (E) numbers of CD45.2+ BM mature B cells (B220+lgM+lgD+), (F) numbers of CD45.2+ BM immature B cells (B220+lgM+lgD−), (G) numbers of CD45.2+ BM Pre B (B220+lgM−lgD−CD43−CD24high), Pro B (B220+lgM−lgD−CD43+CD24med) and Pre-Pro B cells (B220+lgM−lgD−CD43+CD24−), (H) number of peripheral blood CD45.2+CD3+ T cells and (I) number of peripheral blood CD45.2+B220+ B cells was determined by flow cytometry. Absolute number of each cell population was obtained by multiplying the percentage with cellularity of thymus or spleen or WBC count. N=4-6 mice/group. Student t test, * indicates p<0.05 between Pak2-KO and WT. Representative data from 2-3 experiments.

Article Snippet: 23 Primary antibody was rabbit anti-Pak2 polyclonal antibody (Cell signaling Technology, Danvers, MA).

Techniques: Flow Cytometry

Journal: Cell cycle (Georgetown, Tex.)

Article Title: c-MYC protein is degraded in response to UV irradiation.

doi: 10.4161/cc.7.1.5111

Figure Lengend Snippet: Figure 3. Analysis of c-MYC protein domains implicated in its UV-induced degradation pathway. (A) Map of c-MYC domains and of the various mutants constructed. (B) MRC5-SV cells were transfected with pcDNA3 plasmids expressing the indicated FLAG-tagged c-MYC mutants, irradiated with 50 J/m2 UV-C light and post-incubated one hour. Exogenous MYC level was analysed in whole cell extracts by immunoblotting with the anti-FLAG antibody. (C) MRC5-SV cells were transfected with control (Ctrl) or anti-PAK2 siRNAs (1, 2 or 1 and 2 together) at a total 40 nM concentration. After a 48 hours, cells were treated with 50 J/m2 UV-C light and post-incubated for one hour. (D) MRC5-SV cells were trans- fected with pcDNA3 plasmid expressing the indicated FLAG-tagged c-MYC mutants, irradiated with 50 J/m2 UVC light and post-incubated one hour. Exogenous c-MYC level was analysed in whole cell extract by immunoblotting with the anti-FLAG antibody.

Article Snippet: The rabbit monoclonal anti-phospho-S345CHK1 (133D3), the mouse monoclonal anti-CHK1 (2G1D5) and the rabbit polyclonal anti-PAK2 antibodies were from Cell signalling Technology.

Techniques: Construct, Transfection, Expressing, Irradiation, Incubation, Western Blot, Control, Concentration Assay, Plasmid Preparation