p pak4  (Bioss)

Journal: Journal of Translational Medicine

Article Title: Sinensetin serves as an AMPK activator to inhibit RANKL-induced osteoclastogenesis via osteoclast cytoskeleton reorganization

doi: 10.1186/s12967-025-06708-8

Figure Lengend Snippet: SIN prevents the cytoskeleton reorganization of preosteoclasts. a Schematic diagram of osteoclast differentiation and maturation. b Images of crystalline violet staining for the transwell migration assay were recorded. c Statistical area ratio of invaded cells. d Images of cells at the same scratch location after 0 and 24 h in the presence and absence of SIN. e The number of cells in the same region were counted and quantified the difference in number between 0 and 24 h. f Representative images of western blot showed the expression of c-Src, Integrin β3 and Cortactin standardized to β-actin. g – i Quantitative analysis of c-Src, Integrin β3 and Cortactin normalized to β-actin (n = 3). j Representative images of western blot showed the expression of p-PAK4, p-PI3K and p-AKT individually standardized to β-actin, PI3K and AKT. (K-M) Quantitative analysis of p-PAK4, p-PI3K and p-AKT proteins expression (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001. All data are expressed as mean ± SD

Article Snippet: Primary antibodies against c-Fos (ab134122), ATP6V0D2 (ab236375), p-AMPK (ab133448) and AMPK (ab32047) were obtained from Abcam (Cambridge, UK), NFATc1 (sc-7294) and CTSK (sc-48353) were from Santa Cruz Biotechnology (Dallas, USA), p-PAK4 (bs-2270R) were obtained from Bioss (Beijing, China), Cortactin ( R23963 ) were obtained from ZEN-BIOSCIENCE (Chengdu, Sichuan, China), while Nrf2 (16396-1-AP) and Keap1 (10503-2-AP) were form Proteintech Group (Wuhan, Hubei, China).

Techniques: Staining, Transwell Migration Assay, Western Blot, Expressing

Journal: Journal of Translational Medicine

Article Title: Sinensetin serves as an AMPK activator to inhibit RANKL-induced osteoclastogenesis via osteoclast cytoskeleton reorganization

doi: 10.1186/s12967-025-06708-8

Figure Lengend Snippet: SIN inhibits osteoclastogenesis associated with AMPK activity in vivo. a Histological analysis of tibia with TRAcP staining (n = 6 per group). b , c Quantitative analysis of N.Oc/BS and Oc.S/BS of TRAcP-positive cells in tibia. d Representative images of western blot showed the expression of bone proteins including CTSK and ATP6V0D2 standardized to β-actin. e , f Quantitative analysis of CTSK and ATP6V0D2 normalized to β-actin (n = 3). g , i Histological analysis of tibia with p-PAK4 and p-AMPK staining (n = 6 per group). h , j Quantitative analysis of staining area in tibia bone tissue. * p < 0.05, ** p < 0.01, *** p < 0.001. All data are expressed as mean ± SD

Article Snippet: Primary antibodies against c-Fos (ab134122), ATP6V0D2 (ab236375), p-AMPK (ab133448) and AMPK (ab32047) were obtained from Abcam (Cambridge, UK), NFATc1 (sc-7294) and CTSK (sc-48353) were from Santa Cruz Biotechnology (Dallas, USA), p-PAK4 (bs-2270R) were obtained from Bioss (Beijing, China), Cortactin ( R23963 ) were obtained from ZEN-BIOSCIENCE (Chengdu, Sichuan, China), while Nrf2 (16396-1-AP) and Keap1 (10503-2-AP) were form Proteintech Group (Wuhan, Hubei, China).

Techniques: Activity Assay, In Vivo, Staining, Western Blot, Expressing

Journal: Frontiers in Physiology

Article Title: Cofilin activation in pancreatic acinar cells plays a pivotal convergent role for mediating CCK-stimulated enzyme secretion and growth

doi: 10.3389/fphys.2023.1147572

Figure Lengend Snippet: Effect of two PAK4 inhibitors, PF-3758309 or LCH-7749944, on the ability of CCK-8 (0.3 and 100 nM) or TPA (1 µM) to alter activation of cofilin. Isolated pancreatic acini were incubated in the absence or presence of PF-3758309 (0.1 nM) or LCH-7749944 (30 µM) for 3 h and then incubated with no additions (control), CCK-8 (0.3 and 100 nM) for 3 min or TPA (1 µM) for 5 min, and then lysed. Western blots were analyzed using anti-pS3 cofilin. Bands were visualized using chemiluminescence and quantified by densitometry. Top : Results of a representative blot of four independent experiments are shown. Bottom : Means ± S.E. of at least 4 independent experiments. Results are expressed as % of basal stimulation of the control group. *, p < 0.05 compared to the control group; ∞, p < 0.05 compared to stimulants without inhibitors; N.S. , No significant.

Article Snippet: Sennoside A (SE), PAK4 (P-21), anti-goat-HRP-conjugate antibodies, calyculin A and cofilin antibody were from Santa Cruz Biotechnology, Inc. (Dallas, TX).

Techniques: CCK-8 Assay, Activation Assay, Isolation, Incubation, Control, Western Blot

Journal: Frontiers in Physiology

Article Title: Cofilin activation in pancreatic acinar cells plays a pivotal convergent role for mediating CCK-stimulated enzyme secretion and growth

doi: 10.3389/fphys.2023.1147572

Figure Lengend Snippet: Schematic diagram of signaling cascade for activation of cofilin in pancreatic acinar cells. In rat pancreatic acinar cells, maximal activation of cofilin by cholecystokinin (CCK)-8 requires activation of PKC, which mediates both Src family of kinase (SFK) and protein kinase D (PKD) activation resulting in PAK4 activation; requires activation of ROCK, which is mediated by Rho; requires activation of JNK, mediated by activation of this MAPK pathway; and requires activation of serine protein phosphatases (PP2A), which are a substrate of PKC. Cofilin activation is important for CCK-stimulated enzyme secretion as well as ERK1/2 activation which has been shown to mediate growth. Squares represent signaling pathways shown to be involved in cofilin activation in this study.

Article Snippet: Sennoside A (SE), PAK4 (P-21), anti-goat-HRP-conjugate antibodies, calyculin A and cofilin antibody were from Santa Cruz Biotechnology, Inc. (Dallas, TX).

Techniques: Activation Assay, CCK-8 Assay, Protein-Protein interactions

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Targeting p21-activated kinase 4 (PAK4) with pyrazolo[3,4- d ]pyrimidine derivative SPA7012 attenuates hepatic ischaemia-reperfusion injury in mice

doi: 10.1080/14756366.2022.2106478

Figure Lengend Snippet: Drug design and identification of SPA7012 as PAK4 inhibitor. (A) Design strategy for novel PAK4 selective inhibitors. (B) Enzymatic IC 50 curves for SPA7012, SPA7016 and SPA7017. (C) Representative structure of pyrido[3,4- d ]pyrimidine derivatives. (D) Predicted binding mode of SPA7012 (PDB code: 4O0V). Original ligand GNE-2861 (green) and SPA7012 (brown) are presented in the stick model. The hydrogen bonds are indicated as a green dashed line.

Article Snippet: After blocking with 5% skim milk, blots were probed with primary antibodies against: PAK4 (G222), p-PAK4 (S474), p-IKKβ (S176/180), p-IκBα (S32), p-p65 (S536), cleaved caspase-3 (Asp175), Bax (Cell Signalling Technology, Beverly, MA, USA), GAPDH (A531), lamin B1(L75), Bcl2(P65), NQO1 (F252) (Bioworld Technology, St Louis Park, MN, USA), NF-κB p65 (C-20), IKBα (H-4), IKKβ (10A9B6) (Santa Cruz Biotechnology, Dallas, TX, USA), Nrf2 (Proteintech, Rosemont, IL, USA), HO-1 (Enzo Life Sciences, Farmingdale, New York, USA), and p-Thr (Merck KGaA, Darmstadt, Germany).

Techniques: Binding Assay

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Targeting p21-activated kinase 4 (PAK4) with pyrazolo[3,4- d ]pyrimidine derivative SPA7012 attenuates hepatic ischaemia-reperfusion injury in mice

doi: 10.1080/14756366.2022.2106478

Figure Lengend Snippet: PAK4 enzyme inhibitory activity and calculated properties of synthesised compounds

Article Snippet: After blocking with 5% skim milk, blots were probed with primary antibodies against: PAK4 (G222), p-PAK4 (S474), p-IKKβ (S176/180), p-IκBα (S32), p-p65 (S536), cleaved caspase-3 (Asp175), Bax (Cell Signalling Technology, Beverly, MA, USA), GAPDH (A531), lamin B1(L75), Bcl2(P65), NQO1 (F252) (Bioworld Technology, St Louis Park, MN, USA), NF-κB p65 (C-20), IKBα (H-4), IKKβ (10A9B6) (Santa Cruz Biotechnology, Dallas, TX, USA), Nrf2 (Proteintech, Rosemont, IL, USA), HO-1 (Enzo Life Sciences, Farmingdale, New York, USA), and p-Thr (Merck KGaA, Darmstadt, Germany).

Techniques: Activity Assay, Enzyme Inhibition Assay

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Targeting p21-activated kinase 4 (PAK4) with pyrazolo[3,4- d ]pyrimidine derivative SPA7012 attenuates hepatic ischaemia-reperfusion injury in mice

doi: 10.1080/14756366.2022.2106478

Figure Lengend Snippet: PAK subtype selectivity of SPA7012

Article Snippet: After blocking with 5% skim milk, blots were probed with primary antibodies against: PAK4 (G222), p-PAK4 (S474), p-IKKβ (S176/180), p-IκBα (S32), p-p65 (S536), cleaved caspase-3 (Asp175), Bax (Cell Signalling Technology, Beverly, MA, USA), GAPDH (A531), lamin B1(L75), Bcl2(P65), NQO1 (F252) (Bioworld Technology, St Louis Park, MN, USA), NF-κB p65 (C-20), IKBα (H-4), IKKβ (10A9B6) (Santa Cruz Biotechnology, Dallas, TX, USA), Nrf2 (Proteintech, Rosemont, IL, USA), HO-1 (Enzo Life Sciences, Farmingdale, New York, USA), and p-Thr (Merck KGaA, Darmstadt, Germany).

Techniques: Activity Assay

Journal: British Journal of Cancer

Article Title: Nuplazid suppresses esophageal squamous cell carcinoma growth in vitro and in vivo by targeting PAK4

doi: 10.1038/s41416-021-01651-z

Figure Lengend Snippet: a Volcano plot shows that 572 unique phosphosites changed significantly ( t -test P -value < 0.05, FDR < 0.01 upon 24 h treatment with 5 µM Nuplazid). Blue dots represent downregulated proteins, and red dots represent upregulated proteins, with 283 sites up and 289 sites down. b These phosphoproteins which were downregulated are mapped to Heat map upon Nuplazid treatment. c KEGG pathways that are significantly downregulated in phosphorylation are shown according to P -value. d Averaged quantitative phosphosites from the phosphoproteomic, the locations of MAPK1 Y187 were ranked according to their Log2 FC between the DMSO and Nuplazid-treated cells. e Effect of Nuplazid treatment for 24 h on p-MAPK1 T185/Y187 in KYSE150 and KYSE450 cells.

Article Snippet: Antibodies to detect PAK4, p-PAK4 (Ser474) and MAPK1 were purchased from CST (Beverly, MA, USA).

Techniques: Phospho-proteomics

Journal: British Journal of Cancer

Article Title: Nuplazid suppresses esophageal squamous cell carcinoma growth in vitro and in vivo by targeting PAK4

doi: 10.1038/s41416-021-01651-z

Figure Lengend Snippet: a PAK4 kinase activity was assessed by an in vitro kinase assay using active PAK4 and inactive MAPK1 proteins. The effect of Nuplazid was determined by western blot analysis using a p-MAPK1 T185/Y187 antibody. b Nuplazid binds with PAK4 in an ATP-competitive manner. Active PAK4 was incubated with ATP at different concentrations (10, 20 or 100 µM) and 100 µl of Nuplazid–sepharose 4B or sepharose 4B (as a negative control) in reaction buffer. The level of p-PAK4, PAK4, p-MAPK1 and T-MAPK1 in KYSE150 cells ( c ) and KYSE450 cells ( d ) with different concentration of Nuplazid (0, 0.5, 1, 2.5 and 5 μM) treatment for 24 h was determined by western blotting. The level of p-MAPK1 T185/Y187 was affected by PAK4 in KYSE150 cells ( e ) and KYSE450 cells ( f ) which treated with Nuplazid. MAPK1 was immunoprecipitated by PAK4 and MAPK1 was detected by p-MAPK1 T185/Y187 .

Article Snippet: Antibodies to detect PAK4, p-PAK4 (Ser474) and MAPK1 were purchased from CST (Beverly, MA, USA).

Techniques: Activity Assay, In Vitro, Kinase Assay, Western Blot, Incubation, Negative Control, Concentration Assay, Immunoprecipitation

Journal: British Journal of Cancer

Article Title: Nuplazid suppresses esophageal squamous cell carcinoma growth in vitro and in vivo by targeting PAK4

doi: 10.1038/s41416-021-01651-z

Figure Lengend Snippet: a The knockdown efficiency of PAK4 by shRNA (#2, #5) in KYSE150 and KYSE450 cells was evaluated by Immunoblotting. And the level of the PAK4 downstream signal MAPK in the shPAK4 cells was detected by WB. b Cell numbers of KYSE150 and KYSE450 cells with transfected shPAK4 at 0, 24, 48 and 72 h. c Colonies were counted for KYSE150 and KYSE450 cells with transfected with shPAK4 after 7 days. d The overexpression efficiency of PAK4 in KYSE410 cells was evaluated by WB and the level of the PAK4 downstream signal MAPK1 in the KYSE410 cells with transfected PAK4 was detected by WB. e Cell numbers of KYSE410 cells with transfected PAK4 at 0, 24, 48 and 72 h. f Colonies were counted for KYSE410 cells with transfected PAK4 after 7 days. g The KYSE150 cells and KYSE450 cells were treated with 2.5 μM Nuplazid for 96 h, and the inhibition rate was calculated. All data are shown as means ± S.D. The asterisks ( * , ** , *** ) indicate a significant decrease ( p < 0.05, p < 0.01, p < 0.001, respectively).

Article Snippet: Antibodies to detect PAK4, p-PAK4 (Ser474) and MAPK1 were purchased from CST (Beverly, MA, USA).

Techniques: Knockdown, shRNA, Western Blot, Transfection, Over Expression, Inhibition

Journal: British Journal of Cancer

Article Title: Nuplazid suppresses esophageal squamous cell carcinoma growth in vitro and in vivo by targeting PAK4

doi: 10.1038/s41416-021-01651-z

Figure Lengend Snippet: a The photograph showed tumour tissues from PDX mice treated with solvent or Nuplazid (11 mg/kg or 44 mg/kg). b Tumour volumes were measured every 3 days. c After sacrificing, isolated tumours were weighted. d Data recorded tumour size of individual mice. Immunohistochemistry analysed the level of ki67 ( e ) and p-MAPK1 T185/Y187 ( f ) in tumour tissues from treated or untreated groups of mice. All data are shown as means ± S.D. The asterisks ( * , ** , *** ) indicate a significant decrease ( p < 0.05, p < 0.01, p < 0.001, respectively).

Article Snippet: Antibodies to detect PAK4, p-PAK4 (Ser474) and MAPK1 were purchased from CST (Beverly, MA, USA).

Techniques: Solvent, Isolation, Immunohistochemistry