phospho perk  (Cell Signaling Technology Inc)

Journal: Free radical biology & medicine

Article Title: Redox regulation of cell migration via Nischarin S-glutathionylation

doi: 10.1016/j.freeradbiomed.2025.11.013

Figure Lengend Snippet: ( a ) NISCH domain and its interacting partners. NISCH domains interacting with protein partners were drawn in lines in different colors. ( b-d ) Analysis of NISCH-protein interactions and Rac1 downstream pathway in MDA-MB-231 cells. MDA-MB-231 cells expressing NISCH WT or C185S were incubated in low or high glucose (LG or HG). ( b ) NISCH glutathionylation dissociates NISCH from Rac1 and PAK1. After incubating cells in LG or HG for 16 h, the co-immunoprecipitation was used to examine the binding of Rac1, PAK1, or integrin α5 (n = 2–3). ( c ) Rac1 activation analysis after NISCH C185 glutathionylation. After incubating cells for 16 h, the GST-fused p21-binding domain (PBD) derived from PAK1 (GST-PAK1-PBD) was used to pull down the Rac1-GTP form, which was then analyzed by Western blot (n = 2). ( d ) The co-localization analysis of NISCH and Rac1. After 16 h, cells were fixed and analyzed by antibodies to FLAG (green) and Rac1/Cdc42 (red) (n = 10 images). The merged images were analyzed to examine the relative displacement of NISCH and Rac1 in the cell periphery at higher magnification (box). Data represent the mean ± SD. The statistical difference was analyzed by one-way ANOVA and Tukey’s post-hoc test ( b-d ), where *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001.

Article Snippet: The membrane was blocked with 5 % BSA in TBST containing 50 mM Tris-HCl, 150 mM NaCl, and 0.1 % Tween-20, and incubated with primary antibody solution, containing phospho-PAK1 (p423) (1:1000; Cell Signaling, Cat# 2601S), PAK1 (1:1000; Cell Signaling, Cat# 2608S), phospho-cofilin-Ser-3 (Cell Signaling, Cat# 3311S), Cofilin (Cell Signaling, Cat# 3318S), phospho-LIMK-1 (Cell Signaling, Cat# 3841S), LIMK1 (Cell Signaling, Cat# 3842S), phosphor-paxillin (Cell Signaling, Cat# 441026G), Paxillin (Cell Signaling, Cat# 2542S) NISCH (D6T4X) (Cell Signaling, Cat# 85124S), phospho-p38 MAPK (1:1000; Cell Signaling, Cat# 9211S), p38 MAPK (1:1000; Cell Signaling, Cat# 9212S), phospho-MEK1/2 (1:1000; Cell Signaling, Cat# 9154S), MEK1/2 (1:1000; Cell Signaling, Cat# 9122S), phospho-p44/42 MAPK (ERK1/2) (1:1000; Cell Signaling, Cat# 9101S), p44/42 MAPK (ERK1/2) (1:1000; Cell Signaling, Cat# 9102S), phospho-JNK (1:1000; Cell Signaling, Cat# 9251S), JNK (1:1000; Cell Signaling, Cat# 9252S), phospho-p38 (1:1000; Cell Signaling, Cat# 9211S), p38 (1:1000; Cell Signaling, Cat# 9212S), Integrin α−5 (Cell Signaling, Cat# 4705S), GSTO1/2 (1:1000; Santa Cruz, Cat# sc-166040), GSTP (1:1000; MBL Life Science, Cat# 311-H), Glrx3 (1:1000; Sigma, Cat# SAB1410104), or FLAG antibody (1:1000; Millipore-Sigma, Cat# F3165), diluted in a blocking buffer and incubated for overnight at 4°C.

Techniques: Expressing, Incubation, Immunoprecipitation, Binding Assay, Activation Assay, Derivative Assay, Western Blot

Journal: Free radical biology & medicine

Article Title: Redox regulation of cell migration via Nischarin S-glutathionylation

doi: 10.1016/j.freeradbiomed.2025.11.013

Figure Lengend Snippet: MDA-MB-231 cells expressing NISCH WT or C185S were incubated in low or high glucose (LG or HG) for 16 h. ( a ) Phosphorylation levels of PAK1, ERK1/2, and MEK1/2 (n = 3). ( b ) Phosphorylation levels of LIMK1 and cofilin (n = 3). ( c ) Paxillin phosphorylation level (n = 3). ( d ) A model for cell migration induced by NISCH glutathionylation. In a non-stressed condition, NISCH binds to its protein partners, including Rac1 and PAK1, thus suppressing Rac1-and PAK1-mediated cell migration. NISCH may bind and retain Rac1 and integrin-α5 in the cytosol or endosome. However, upon ROS production, NISCH is glutathionylated at C185, dissociating Rac1 and PAK1, but not integrin-α5. The activated PAK1 activates its downstream LIMK1 and inhibits cofilin through phosphorylation, thereby increasing actin dynamics and polymerization. The dissociated Rac1 increases its localization to the membrane, where it activates actin filament branching, induces lamellipodia and membrane ruffles, and increases cell migration. Data represent the mean ± SD. The statistical difference was analyzed by one-way ANOVA and Tukey’s post-hoc test ( a – c ), where *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001.

Article Snippet: The membrane was blocked with 5 % BSA in TBST containing 50 mM Tris-HCl, 150 mM NaCl, and 0.1 % Tween-20, and incubated with primary antibody solution, containing phospho-PAK1 (p423) (1:1000; Cell Signaling, Cat# 2601S), PAK1 (1:1000; Cell Signaling, Cat# 2608S), phospho-cofilin-Ser-3 (Cell Signaling, Cat# 3311S), Cofilin (Cell Signaling, Cat# 3318S), phospho-LIMK-1 (Cell Signaling, Cat# 3841S), LIMK1 (Cell Signaling, Cat# 3842S), phosphor-paxillin (Cell Signaling, Cat# 441026G), Paxillin (Cell Signaling, Cat# 2542S) NISCH (D6T4X) (Cell Signaling, Cat# 85124S), phospho-p38 MAPK (1:1000; Cell Signaling, Cat# 9211S), p38 MAPK (1:1000; Cell Signaling, Cat# 9212S), phospho-MEK1/2 (1:1000; Cell Signaling, Cat# 9154S), MEK1/2 (1:1000; Cell Signaling, Cat# 9122S), phospho-p44/42 MAPK (ERK1/2) (1:1000; Cell Signaling, Cat# 9101S), p44/42 MAPK (ERK1/2) (1:1000; Cell Signaling, Cat# 9102S), phospho-JNK (1:1000; Cell Signaling, Cat# 9251S), JNK (1:1000; Cell Signaling, Cat# 9252S), phospho-p38 (1:1000; Cell Signaling, Cat# 9211S), p38 (1:1000; Cell Signaling, Cat# 9212S), Integrin α−5 (Cell Signaling, Cat# 4705S), GSTO1/2 (1:1000; Santa Cruz, Cat# sc-166040), GSTP (1:1000; MBL Life Science, Cat# 311-H), Glrx3 (1:1000; Sigma, Cat# SAB1410104), or FLAG antibody (1:1000; Millipore-Sigma, Cat# F3165), diluted in a blocking buffer and incubated for overnight at 4°C.

Techniques: Expressing, Incubation, Phospho-proteomics, Migration, Membrane

Journal: Life Science Alliance

Article Title: Brain vascular stability relies on PAK2–cilia–PDGF-BB–HSPGs on basolateral side of endothelium

doi: 10.26508/lsa.202503460

Figure Lengend Snippet: (A, B) ELISAs performed on supernatants from control siRNA, PAK2 siRNA (A), and ARL3b siRNA (B) brain ECs with or without heparinase II treatment. (C, D, E) Immunofluorescent brain images of e11.5 WT Ext1 fl/fl mouse cortex stained for endomucin (green), PDGF-BB (red), and merged image. (F) Immunofluorescence image of PDGF-BB antibody stained (green) in a Tg( flk :mCherry) (red) 52-hpf zebrafish embryo. (F, F′, F″) White arrows point to PHBC. (F, F′, F″) Orange box in panel (F) is magnified in (F′, F″) showing PDGF-BB gradient-like expression close to red blood vessels in the brain. The scale bar in (F) is 100 μm.

Article Snippet: Proteins were transferred to PVDF membranes, blocked in 5% nonfat dry milk in TBST, and probed with the following primary antibodies: ARL13B (Cat# 17711-1-AP; Proteintech), PAK2 (Cat# 2608S; Cell Signaling Technology), PDGF-BB (Cat# NBP1-58279; Novus Biologicals), and β-actin (Cat# MA1-744; Thermo Fisher Scientific) as a loading control.

Techniques: Control, Staining, Immunofluorescence, Expressing

Journal: Life Science Alliance

Article Title: Brain vascular stability relies on PAK2–cilia–PDGF-BB–HSPGs on basolateral side of endothelium

doi: 10.26508/lsa.202503460

Figure Lengend Snippet: (A, A′) IF for cilia ARL13b (green) and PDGF-BB (red) in brain microvascular ECs. (A, A′) Boxed region in panel (A) is zoomed in panel (A′). An arrow indicates a brain EC cilium that is colocalized with PDGF-BB. (A, A′) Scale bars: 20 μm for (A) and 10 μm for (A′). (B) Western blot of brain endothelial cell lysates from cells transfected with control or PDGF-BB siRNA. PDGF-BB, ARL13b, PAK2, and β-actin proteins were probed with specific antibodies and blots quantified by ImageJ. (B, C) Quantification data of the Western blots in panel (B). n = 3 in each experimental group. Results are presented as the mean ± SEM. Statistics were performed using paired t tests. P < 0.05 was considered significant. Source data are available for this figure.

Article Snippet: Proteins were transferred to PVDF membranes, blocked in 5% nonfat dry milk in TBST, and probed with the following primary antibodies: ARL13B (Cat# 17711-1-AP; Proteintech), PAK2 (Cat# 2608S; Cell Signaling Technology), PDGF-BB (Cat# NBP1-58279; Novus Biologicals), and β-actin (Cat# MA1-744; Thermo Fisher Scientific) as a loading control.

Techniques: Western Blot, Transfection, Control

Journal: Life Science Alliance

Article Title: Brain vascular stability relies on PAK2–cilia–PDGF-BB–HSPGs on basolateral side of endothelium

doi: 10.26508/lsa.202503460

Figure Lengend Snippet: (A, B) qRT-PCR for indicated HS proteoglycan gene synthesis and modifying enzyme targets in PAK2 siRNA ECs (A) and ARL13b siRNA ECs (B), respectively. (C) HS disaccharide analysis in control and PAK2 siRNA brain ECs. (D) PDGF-BB cell surface ELISA binding assay on control, PAK2 siRNA brain ECs, and ARL13b siRNA brain ECs. Heparinase treatment is used as a positive control. (E, F) Panels are immunofluorescent HSPG antibody stained (green) in pak2a WT (E) or pak2a homozygous bleeder (F) 52-hpf embryo in a Tg( flk :mCherry) (red) fish background. MCeV stands for mid-cerebral vein. The arrow points to region where HSPGs are stained in MCeV, which is surrounding the blood vessel outline. Scale bars in (E, F) are 100 μm. (A, B) T test was used for statistical analysis of qRT-PCR data in (A, B).

Article Snippet: Proteins were transferred to PVDF membranes, blocked in 5% nonfat dry milk in TBST, and probed with the following primary antibodies: ARL13B (Cat# 17711-1-AP; Proteintech), PAK2 (Cat# 2608S; Cell Signaling Technology), PDGF-BB (Cat# NBP1-58279; Novus Biologicals), and β-actin (Cat# MA1-744; Thermo Fisher Scientific) as a loading control.

Techniques: Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Binding Assay, Positive Control, Staining

Journal: Life Science Alliance

Article Title: Brain vascular stability relies on PAK2–cilia–PDGF-BB–HSPGs on basolateral side of endothelium

doi: 10.26508/lsa.202503460

Figure Lengend Snippet: (A, B) Quantification of EC ARL13b cilium numbers (A) and ARL13b cilium length (B) in control, PAK2 siRNA brain ECs, and ARL13b siRNA brain ECs with and without heparinase (H) treatment. (C) Panel shows quantification of the number of EC ARL13b cilia in n = 8 pak2a homozygous bleeders (B) (mi149/mi149), n = 6 pak2a heterozygous non-bleeders (NB) (mi149/WT), and n = 5 pak2a WT (WT/WT). (D, E) Whole-mount images of control vehicle (D) or human PDGF-BB protein (1 ng)–injected zebrafish 52-hpf embryo. (D, E) Note brain bleeding is reduced in panel (E) compared with panel (D). (D, E, F) Quantification of the bleeding area in panels (D, E). N = 6 for control vehicle–injected and N = 5 for PDGFB-BB protein–injected embryos. Scale bars in (D) are 200 μm. (C) GLM with negative binomial distribution was performed to compare the number of cilia among group in (C). Dunnett’s test was used to adjust for multiple comparisons. Estimated means and 95% confidence intervals (CIs) were used for plotting the number of cilia. WT had more cilia compared with group B or NB, adjusted P = 0.0018 and 0.013, respectively. (F) Unpaired t test was used to analyze bleeding data in (F). ** P = 0.0031. (G, H) Immunofluorescent ARL13b antibody–stained (green) images of human PDGF-BB protein (1 ng)–injected (G) or control vehicle–injected (H) Tg( flk :mCherry) rhd mi149/mi149 (homozygous) bleeders. White arrows in (G, H) show Keyence microscope measurement of dimensions of the area analyzed for quantitation of endothelial cilia: 160 μm posterior to the MTA/MCeV and 90 μm dorsal to PHBC. MTA, metencephalic artery; MCeV, middle cerebral vein; PHBC, primordial hindbrain channels. (G, G′, H, H′) White boxes in panels (G, H) are magnified in panels (G′, H′). (G′, H′) Note: Many cilia are seen in panel (G′) (white arrows) compared with panel (H′). Panel (I) is the quantification of the number of endothelial cilia in the human PDGF-BB protein (1 ng)–injected and control vehicle–injected rhd mi149/mi149 (homozygous) bleeders. N = 3 embryos were quantified in each group. Scale bars in (G, G′, H, H′) are 50 μm. An unpaired t test was used to analyze differences in the number of cilia, which were found to be not statistically significant.

Article Snippet: Proteins were transferred to PVDF membranes, blocked in 5% nonfat dry milk in TBST, and probed with the following primary antibodies: ARL13B (Cat# 17711-1-AP; Proteintech), PAK2 (Cat# 2608S; Cell Signaling Technology), PDGF-BB (Cat# NBP1-58279; Novus Biologicals), and β-actin (Cat# MA1-744; Thermo Fisher Scientific) as a loading control.

Techniques: Control, Injection, Staining, Microscopy, Quantitation Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: DOCK10 Regulates Insulin Hypersecretion in Insulinoma and Serves as a Diagnostic and Therapeutic Target

doi: 10.1016/j.jcmgh.2025.101705

Figure Lengend Snippet: DOCK10 regulates insulin secretion via CDC42 in MIN6 cells. ( A ) Dock10 mRNA levels by qPCR in control and DOCK10-knockdown (KD) MIN6 cells. ( B ) ELISA measurement of insulin secretion after stimulation with 3 mM glucose, 25 mM glucose, or 3 mM glucose + 30 mM KCl. ( C ) ELISA measurement of intracellular insulin levels in control and knockdown MIN6 cells (n = 3/group). ( D ) Schematic of Dock10–Cdc42 pathway regulating GSIS; ML141, a Cdc42 inhibitor. ( E ) Protein levels of phosphorylated PAK1 (p-PAK1), total PAK1, and β-actin after glucose stimulation (2-hour fasting) in control and knockdown cells by Simple Western; table shows p-PAK1/PAK1 ratio normalized to β-actin. ( F ) Protein levels after 1-hour fasting and ML141 or vehicle (DMSO) pretreatment, assessed by Simple Western; table shows normalized p-PAK1/PAK1 ratios. ( G ) ELISA measurement of insulin secretion after ML141 or vehicle pretreatment under the same stimulations (n = 3/group). Data were pooled from 3 independent experiments for panels in ( A–C ) and ( G ). Values are presented as mean ± SD. Statistical analysis was performed using unpaired Student’s t -test. ns, not significant; ∗∗ P < .01; ∗∗∗ P < .005.

Article Snippet: phospho PAK1 , Cell Signaling Technology , 2601S , 1:10.

Techniques: Control, Knockdown, Enzyme-linked Immunosorbent Assay, Simple Western