rabbit polyclonal anti-pak2  (Cell Signaling Technology Inc)

Journal: Redox Biology

Article Title: Changes to insulin sensitivity in glucose clearance systems and redox following dietary supplementation with a novel cysteine-rich protein: A pilot randomized controlled trial in humans with type-2 diabetes

doi: 10.1016/j.redox.2023.102918

Figure Lengend Snippet: The effect of 14-weeks of CON, WHEY or KDPWHE treatment in adults with type-2 diabetes mellitus on phosphoprotein concentration within the insulin-receptor signalling pathway upstream of regulatory nodes for GLUT4 translocation within the skeletal muscle in responses to basal (INS -) and insulin stimulation (hyperinsulinemic-isoglycaemic; INS +), with data shown as relative protein abundance at week 0 and 15. (A) Insulin-receptor substrate-1 phosphorylated on Ser312 (IRS-1 Ser312 ). (B) Representative immunofluorescence images of cross-sectional skeletal muscle fibres stained for IRS-1 Ser312 . IRS-1 ser312 was measured as staining intensity in the plasma membrane region (SM ). The small grey scale lines bottom right represent 50 μm. (C) Protein kinase B phosphorylation on Ser437 (Akt Ser437 ) expressed relative to total Akt. (D) Akt substrate 160 kDa phosphorylation on Thr642 (AS160 Thr642 ), (E) p21 activated kinase (PAK) phosphorylation on isoform 1 (PAK Thr423 ) and 2 (PAK Thr402 ). Data are raw unit median, upper and lower quartiles, and range, with individual-participant data points included. Raw unit point and change score mean and SD are in SM Data 1. Outcome statistics are in . CON, non-protein isocaloric control; KDPWHE, keratin-derived protein with whey; WHEY, whey protein isolate.

Article Snippet: Blocked membranes were then incubated overnight at 4 °C with the following antibodies diluted in TBS-T/3%BSA; rabbit polyclonal anti-human PRX2 at 1:10,000 (R8656, Sigma-Aldrich, RRID: AB_1841096 ), rabbit polyclonal anti-human PRX3 at 1:10,000 (PA0030 AbFrontier, RRID: AB_1620980 ), rabbit monoclonal anti-NRF2 at 1:1, 000 (ab62352, Abcam, RRID: AB_944418 ), rabbit polyclonal anti-GPx1 at 1:1000 (11797, ThermoFisher, RRID: AB_2723218 ), mouse monoclonal anti-SOD1 at 1:1000 (MA1-105, ThermoFisher, RRID: AB_2536811 ), rabbit polyclonal anti-phospho-AS160 Thr642 at 1:1000 (4288, Cell Signalling Technology), rabbit polyclonal anti-phospho-PAK1 Thr423 /p-PAK2 Thr402 at 1:1000 (2601, Cell Signalling Technology), rabbit polyclonal anti-phospho-Akt Ser437 at 1:1000 (44-621G, ThermoFisher), rabbit monoclonal anti-Pan-Akt at 1:1000 (MA5-14916, Thermofisher), mouse monoclonal anti-α-tubulin HRP-conjugate at 1:1000 (ab40742, Abcam).

Techniques: Concentration Assay, Translocation Assay, Quantitative Proteomics, Immunofluorescence, Staining, Clinical Proteomics, Membrane, Phospho-proteomics, Control, Derivative Assay

Journal: Redox Biology

Article Title: Changes to insulin sensitivity in glucose clearance systems and redox following dietary supplementation with a novel cysteine-rich protein: A pilot randomized controlled trial in humans with type-2 diabetes

doi: 10.1016/j.redox.2023.102918

Figure Lengend Snippet: The effect of 14-weeks of KDPWHE, WHEY, or CON treatment in adults with type-2 diabetes mellitus on skeletal muscle tissue insulin signalling, capillarization, and capillary endothelial nitric oxide synthase activity.

Article Snippet: Blocked membranes were then incubated overnight at 4 °C with the following antibodies diluted in TBS-T/3%BSA; rabbit polyclonal anti-human PRX2 at 1:10,000 (R8656, Sigma-Aldrich, RRID: AB_1841096 ), rabbit polyclonal anti-human PRX3 at 1:10,000 (PA0030 AbFrontier, RRID: AB_1620980 ), rabbit monoclonal anti-NRF2 at 1:1, 000 (ab62352, Abcam, RRID: AB_944418 ), rabbit polyclonal anti-GPx1 at 1:1000 (11797, ThermoFisher, RRID: AB_2723218 ), mouse monoclonal anti-SOD1 at 1:1000 (MA1-105, ThermoFisher, RRID: AB_2536811 ), rabbit polyclonal anti-phospho-AS160 Thr642 at 1:1000 (4288, Cell Signalling Technology), rabbit polyclonal anti-phospho-PAK1 Thr423 /p-PAK2 Thr402 at 1:1000 (2601, Cell Signalling Technology), rabbit polyclonal anti-phospho-Akt Ser437 at 1:1000 (44-621G, ThermoFisher), rabbit monoclonal anti-Pan-Akt at 1:1000 (MA5-14916, Thermofisher), mouse monoclonal anti-α-tubulin HRP-conjugate at 1:1000 (ab40742, Abcam).

Techniques: Activity Assay, Phospho-proteomics

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: Molecular psychiatry

Article Title: Phosphoproteomic of the acetylcholine pathway enables discovery of the PKC-β-PIX-Rac1-PAK cascade as a stimulatory signal for aversive learning.

doi: 10.1038/s41380-022-01643-2

Figure Lengend Snippet: Fig. 3 The cholinesterase inhibitor donepezil activates the M1R-PKC-PAK cascade in accumbal D2R-MSNs in vivo. A C57BL/6J mice were preadministered the M1R antagonist VU0255035 [40 mg/kg, intraperitoneal (i.p.)] 25 min before donepezil administration [0.4 mg/kg, subcutaneous (s.c.)]. Thirty minutes after donepezil administration, the mice were subjected to immunoblotting analysis. n = 6. The error bars represent the mean ± SEM. One-way ANOVA was followed by Tukey’s test, *p < 0.05, **p < 0.01, ***p < 0.001. B Drd1a-tdTomato/Drd2-mVenus double transgenic mice were preadministered the M1R antagonist VU0255035 (40 mg/kg, i.p.) 25 min before donepezil administration (0.4 mg/kg, s.c.). Thirty minutes after donepezil administration, the mice were subjected to immunohistochemical analysis. The striatal/ accumbal slices (25 μm) were stained with anti-pPAK1 (S144) and anti-GFP antibodies. Nuclei were visualized with TO-PRO3. Arrows indicate pPAK1 (S144)-positive D2R-MSNs. Scale bar, 10 μm. C The data plot shows the percentage of D1R-MSNs or D2R-MSNs among pPAK1 (S144)- positive cells when mice were administered with donepezil (0.4 mg/kg, s.c.). The error bars represent the mean ± SEM of three independent experiments. Student’s t test, **p < 0.01. D Quantification of the pPAK1 (S144)-positive D2R-MSNs in the NAc core. The error bars represent the mean ± SEM of three independent experiments. One-way ANOVA followed by Tukey’s test, ***p < 0.001.

Article Snippet: The following antibodies were used for immunoblotting: rabbit polyclonal anti-phospho PAK1 (S144) (RRID:AB_2299279, #2606S, 1:1000), rabbit polyclonal anti-phospho NMDAR1 (S890) (RRID:AB_2294781, #3381S, 1:1000), rabbit polyclonal anti-PAK1 (RRID:AB_330222, #2602S, 1:1000), rabbit monoclonal anti-phospho cofilin (S3) (RRID: AB_2080597, #3313S, 1:1000) (Cell Signaling Technology, Inc, Danvers, MA, USA); mouse monoclonal anti-cofilin (RRID:AB_11043339, #66057-1-Ig, 1:1000) (Proteintech, Rosemont, IL, USA); mouse monoclonal anti-LIMK1 (RRID:AB_ 2723320, #MA5-25569, 1:1000) (Thermo Fisher Scientific, Waltham, MA, USA); rabbit polyclonal anti-phospho LIMK1 (T508) (RRID:AB_10621739, #SAB4300103, 1:1000) (Sigma-Aldrich); mouse monoclonal anti-NMDAR1 (RRID:AB_10002447, #NB300-118, 1:2000) (Novus Biologicals, Littleton, CO, USA); mouse monoclonal anti-GST (RRID: AB_2883970, #017-21854, 1:1000), mouse monoclonal anti-β-Actin (RRID: AB_2858279, #010-27841, 1:2000) (Fujifilm Wako, Osaka, Japan); mouse monoclonal anti-BetaPIX (RRID:AB_399166, # 611648, 1:1000) (BD Biosciences, San Jose, CA, USA); rabbit polyclonal anti-phospho BetaPIX (T76), (1:500) (kindly provided by NS, Kobe University, Kobe, Japan); goat anti-rabbit IgG Alexa Fluor 680 (RRID:AB_2535758, #A-21109, 1:20,000) (Thermo Fisher Scientific); and goat anti-mouse IgG DyLight conjugate 800 (RRID:AB_10693543, #5257P, 1:20,000) (Cell Signaling Technology).

Techniques: In Vivo, Western Blot, Transgenic Assay, Immunohistochemical staining, Staining

Journal: Molecular psychiatry

Article Title: Phosphoproteomic of the acetylcholine pathway enables discovery of the PKC-β-PIX-Rac1-PAK cascade as a stimulatory signal for aversive learning.

doi: 10.1038/s41380-022-01643-2

Figure Lengend Snippet: Fig. 4 Electric foot shock activates PAK through M1Rs in accumbal D2R-MSNs. A VU0255035 was administered i.p. 25 min before the electric foot shock (0.4 mA, 2.0 s). n = 10. Error bars indicate mean ± SEM. Kruskal–Wallis test, followed by Dunn’s multiple comparisons test, *p < 0.05, **p < 0.01. B FRAX486 was administered s.c. 6 h before the electric foot shock (0.4 mA, 2.0 s). n = 8. Error bars indicate mean ± SEM. Kruskal–Wallis test, followed by Dunn’s multiple comparisons test, n.s. not significant, **p < 0.01. C VU0255035 was administered (40 mg/kg, i.p.) before the electric foot shock (0.4 mA, 2.0 s). Ten minutes after electric foot shock, NR1 phosphorylation and PAK autophosphorylation were quantified by immunoblotting. n = 7. Error bars indicate mean ± SEM. One-way ANOVA followed by Tukey’s test, ***p < 0.001, ****p < 0.0001. D Drd1a-tdTomato/Drd2-mVenus double transgenic mice were administered with VU0255035 (40 mg/kg, i.p.) before the electric foot shock. Ten minutes after electric foot shock, immunohistochemical analysis was performed. Immunofluorescence staining using an anti-pPAK1 (S144) antibody, an anti-GFP antibody, and TO-PRO3 is shown. Arrows indicate pPAK1 (S144)-positive D2R-MSNs. Scale bar, 10 μm. E The percentage of D1R-MSNs or D2R-MSNs among pPAK1 (S144)-positive cells after electric foot shock. Error bars indicate the mean ± SEM of three independent experiments. Student’s t test, **p < 0.01. F Quantification of the pPAK1 (S144)-positive D2R-MSNs in the NAc core. Error bars indicate the mean ± SEM of three independent experiments. One-way ANOVA followed by Tukey’s test, ****p < 0.0001.

Article Snippet: The following antibodies were used for immunoblotting: rabbit polyclonal anti-phospho PAK1 (S144) (RRID:AB_2299279, #2606S, 1:1000), rabbit polyclonal anti-phospho NMDAR1 (S890) (RRID:AB_2294781, #3381S, 1:1000), rabbit polyclonal anti-PAK1 (RRID:AB_330222, #2602S, 1:1000), rabbit monoclonal anti-phospho cofilin (S3) (RRID: AB_2080597, #3313S, 1:1000) (Cell Signaling Technology, Inc, Danvers, MA, USA); mouse monoclonal anti-cofilin (RRID:AB_11043339, #66057-1-Ig, 1:1000) (Proteintech, Rosemont, IL, USA); mouse monoclonal anti-LIMK1 (RRID:AB_ 2723320, #MA5-25569, 1:1000) (Thermo Fisher Scientific, Waltham, MA, USA); rabbit polyclonal anti-phospho LIMK1 (T508) (RRID:AB_10621739, #SAB4300103, 1:1000) (Sigma-Aldrich); mouse monoclonal anti-NMDAR1 (RRID:AB_10002447, #NB300-118, 1:2000) (Novus Biologicals, Littleton, CO, USA); mouse monoclonal anti-GST (RRID: AB_2883970, #017-21854, 1:1000), mouse monoclonal anti-β-Actin (RRID: AB_2858279, #010-27841, 1:2000) (Fujifilm Wako, Osaka, Japan); mouse monoclonal anti-BetaPIX (RRID:AB_399166, # 611648, 1:1000) (BD Biosciences, San Jose, CA, USA); rabbit polyclonal anti-phospho BetaPIX (T76), (1:500) (kindly provided by NS, Kobe University, Kobe, Japan); goat anti-rabbit IgG Alexa Fluor 680 (RRID:AB_2535758, #A-21109, 1:20,000) (Thermo Fisher Scientific); and goat anti-mouse IgG DyLight conjugate 800 (RRID:AB_10693543, #5257P, 1:20,000) (Cell Signaling Technology).

Techniques: Phospho-proteomics, Western Blot, Transgenic Assay, Immunohistochemical staining, Staining