Journal: The Journal of Cell Biology

Article Title: Lack of Paxillin phosphorylation promotes single-cell migration in vivo

doi: 10.1083/jcb.202206078

Figure Lengend Snippet: Paxillin exhibits reduced phosphorylation on Y118 in migrating cancer cells in vivo as compared to in vitro cell culture conditions in both zebrafish and mouse melanoma models. (A) Schematic of protein structures of human and zebrafish Paxillin (top) and amino acid sequence comparisons of the region encompassing Y118 between zebrafish Paxillin and vertebrate Paxillin (bottom). Red arrowhead and box indicate the conservation of Y118 Paxillin between zebrafish and other vertebrates. (B) Top: Endogenous pY118-Paxillin staining (magenta) of ZMEL-GFP (GFP immunostaining, green) plated on 2D in vitro cell culture dishes. White arrowheads mark positive pY118-Paxillin staining. Bottom: Endogenous pY118-Paxillin staining (magenta) of ZMEL-mCherry (mCherry immunostaining, pseudo-colored green, white arrowheads) in larval zebrafish (3 d post-transplantation). Red arrowhead indicates a non-ZMEL cell with positive pY118-Paxillin immunostaining. Zoomed regions reveal pY118-Paxillin immunostaining only. Scale bar is 10 µm. (C) Western blot analysis of mouse melanoma YUMM1.7 cells expressing mammalian WT-Paxillin-T2A-GFP plated on the in vitro cell culture dishes ( n = 3 dishes) and YUMM1.7 melanoma in vivo tumors ( n = 5 tumors). In vitro and in vivo bands are from the same blot—see unmodified Western blot in . GFP was used as the loading control and a control for the number of YUMM1.7 cells in mouse tumors. (D) Quantification of pY118-Paxillin/total Paxillin protein ratio from C. Non-parametric unpaired t -test. (E) Quantification of single cell migration velocity in ZMEL-mCherry cells that exogenously express GFP-tagged zebrafish WT-Paxillin, Y118E-Paxillin, or Y118F-Paxillin in the in vitro cell culture conditions ( n = 64 cells for WT, n = 32 cells for Y118E, and n = 35 cells for Y118F) and in vivo ( n = 8 cells/3 fish for WT, n = 12 cells/3 fish for Y118E, and n = 15 cells/3 fish for Y118F). Larval zebrafish are imaged 1 d post-transplantation. Non-parametric one-way ANOVA, error bars are mean ± SD. (F) Cumulative FRAP recovery curves of WT-Paxillin-EGFP, Y118E-Paxillin-EGFP, or Y118F-Paxillin-EGFP in ZMEL cells in the in vitro cell culture conditions and in vivo after photobleaching. n = 34, 44, and 51 cells for WT, Y118E, Y118F in vitro, and n = 7 cells/6 fish, 6 cells/6 fish, and 6 cells/5 fish for WT, Y118E, Y118F in vivo. (G) Quantification of Paxillin disassembly rates in the WT, Y118E, Y118F-Paxilllin under in vitro cell culture conditions and WT, Y118E, Y118F-Paxilllin under in vivo conditions. n = 13, 13, and 11 cells for WT, Y118E, Y118F in vitro, and n = 8 cells/7 fish, 6 cells/6 fish, 11 cells/10 fish for WT, Y118E, Y118F in vivo. Error bars are mean ± SD. Non-parametric unpaired t test. Source data are available for this figure: .

Article Snippet: Primary antibodies used were rabbit anti-Paxillin (1:1,000, STJ94969; Antibodyplus), rabbit anti-pY118-Paxillin (1:1,000, 9369; Cell Signaling Technology), rabbit anti-FAK (1:1,000, 3285; Cell Signaling Technology), rabbit anti-pFAK397 (1:1,000, 3283; Cell Signaling Technology), chicken anti-GFP (1:500, ab13970; Abcam), mouse anti-CrkII (1:1,000610035; BD Bioscience), mouse anti-DOCK180 (1:500, sc-13163; Santa Cruz Biotechnology), mouse anti-C3G (1:250, sc-178403; Santa Cruz Biotechnology), rabbit p-ERK (1:1,000, 9101S; Cell Signaling Technology), and rabbit anti-pY31-Paxillin (1:1,000, 44-720G; Thermo Fisher Scientific).

Techniques: In Vivo, In Vitro, Cell Culture, Sequencing, Staining, Immunostaining, Transplantation Assay, Western Blot, Expressing, Migration

Journal: The Journal of Cell Biology

Article Title: Lack of Paxillin phosphorylation promotes single-cell migration in vivo

doi: 10.1083/jcb.202206078

Figure Lengend Snippet: Y118-Paxillin exhibits distinct phosphorylation status in migrating cancer cells in vivo versus in vitro. (A) Top: pY118-Paxillin immunostaining (magenta) of ZMEL-GFP (GFP immunostaining, green) plated on in vitro cell culture dishes. Middle: pY118-Paxillin immunostaining (magenta) of ZMEL-mCherry (mCherry immunostaining, pseudo-colored green) in larval zebrafish (3 d post-transplantation). Bottom: pY118-Paxillin immunostaining (magenta) of the zebrafish developing heart (5 dpf). (B) Western blot showing the specificity of the pY118-Paxillin antibody and that it does not recognize Y118E-Paxillin and Y118F-Paxillin. (C) Representative images of ZMEL-GFP cells plated on 2D surfaces of different stiffnesses (left) and stained for pY118-Paxillin (right). (D) Unmodified Western blot of panels shown in —YUMM1.7 cells plated in culture and YUMM1.7 melanoma tumors in vivo blotted with Paxillin and pY118-Paxillin antibodies. “P” is parental cell line with no GFP expression. GFP was used as the loading control and as a control for the number of YUMM1.7 cells in mouse tumors. Source data are available for this figure: .

Article Snippet: Primary antibodies used were rabbit anti-Paxillin (1:1,000, STJ94969; Antibodyplus), rabbit anti-pY118-Paxillin (1:1,000, 9369; Cell Signaling Technology), rabbit anti-FAK (1:1,000, 3285; Cell Signaling Technology), rabbit anti-pFAK397 (1:1,000, 3283; Cell Signaling Technology), chicken anti-GFP (1:500, ab13970; Abcam), mouse anti-CrkII (1:1,000610035; BD Bioscience), mouse anti-DOCK180 (1:500, sc-13163; Santa Cruz Biotechnology), mouse anti-C3G (1:250, sc-178403; Santa Cruz Biotechnology), rabbit p-ERK (1:1,000, 9101S; Cell Signaling Technology), and rabbit anti-pY31-Paxillin (1:1,000, 44-720G; Thermo Fisher Scientific).

Techniques: In Vivo, In Vitro, Immunostaining, Cell Culture, Transplantation Assay, Western Blot, Staining, Expressing

Journal: The Journal of Cell Biology

Article Title: Lack of Paxillin phosphorylation promotes single-cell migration in vivo

doi: 10.1083/jcb.202206078

Figure Lengend Snippet: Macrophages expressing non-phosphorylatable Y118F-Paxillin exhibit increased motility in vivo. (A) Endogenous pY118 Paxillin immunostaining (magenta) of macrophages (green, white arrowheads) in Tg ( mpeg:Lifeact-GFP ) zj506 larval zebrafish. Red arrowhead marks positive pY118 Paxillin immunostaining of a non-macrophage cell. Zoomed region of macrophage lacking pY118-Paxillin immunostaining. (B) Schematic of zebrafish tail wound transection area and macrophage imaging area for directed cell migration. (C) Still images from zebrafish macrophage tracking timelapse videos in 3 dpf Tg ( mpeg:WT-zebrafish Paxillin- EGFP ) zj503 , Tg ( mpeg:zebrafish Y118E-Paxillin- EGFP ) zj504 , and Tg ( mpeg:zebrafish Y118F-Paxillin- EGFP ) zj505 larvae at timepoint 0 and 10 min. Dotted lines indicate wound sites and arrows show the direction of migration. See also . Scale bar is 10 µm. (D) Quantification of macrophage migration velocities toward the wound in vivo. Non-parametric one-way ANOVA, error bars are mean ± SD. n = 38 cells/6 fish for WT, n = 20 cells/6 fish for Y118E and n = 24 cells/10 fish for Y118F. (E) Cell tracking of macrophage migration trajectories toward the wound in vivo, migration starting points are normalized to 0 in both x and y axes, wound sites are normalized to the positive x axis ( n = 38 cells/6 fish for WT, n = 20 cells/6 fish for Y118E and n = 24 cells/10 fish for Y118F). Arrows show the direction of migration toward the wound.

Article Snippet: Primary antibodies used were rabbit anti-Paxillin (1:1,000, STJ94969; Antibodyplus), rabbit anti-pY118-Paxillin (1:1,000, 9369; Cell Signaling Technology), rabbit anti-FAK (1:1,000, 3285; Cell Signaling Technology), rabbit anti-pFAK397 (1:1,000, 3283; Cell Signaling Technology), chicken anti-GFP (1:500, ab13970; Abcam), mouse anti-CrkII (1:1,000610035; BD Bioscience), mouse anti-DOCK180 (1:500, sc-13163; Santa Cruz Biotechnology), mouse anti-C3G (1:250, sc-178403; Santa Cruz Biotechnology), rabbit p-ERK (1:1,000, 9101S; Cell Signaling Technology), and rabbit anti-pY31-Paxillin (1:1,000, 44-720G; Thermo Fisher Scientific).

Techniques: Expressing, In Vivo, Immunostaining, Imaging, Migration, Cell Tracking Assay

Journal: The Journal of Cell Biology

Article Title: Lack of Paxillin phosphorylation promotes single-cell migration in vivo

doi: 10.1083/jcb.202206078

Figure Lengend Snippet: FAK is downregulated and CRKII-DOCK180/RacGEF exhibits increased interaction with unphosphorylated Y118-Paxillin in vivo compared to in vitro. (A) Schematic of in vitro Paxillin regulation from cell culture studies. Following integrin activation, a tyrosine kinase, FAK, phosphorylates Paxillin. Phosphorylated Paxillin then recruits the adaptor protein CRKII and the Paxillin/CRKII complex further recruits DOCK180/RacGEF, thereby activating downstream Rac-dependent pathways, inducing cell migration. (B) Western blot analysis of FAK levels (FAK) and FAK activation (pY397-FAK) in YUMM1.7 cells expressing mammalian WT-Paxillin-T2A-GFP in culture and YUMM1.7 tumors in vivo. In vitro and in vivo bands are from the same blot. Unmodified Western blot is in . GFP was used as the loading control and as a control for the number of YUMM1.7 cells in mouse tumors. (C and D) Quantification of the pY397-FAK/total FAK ratio (C) and total normalized FAK to GFP expression (D) in the in vitro cell culture and in vivo conditions. n = 3 dishes, 5 tumors for C, n = 5 dishes, 8 tumors for D. Error bars are mean ± SD. Non-parametric unpaired t test. (E) Western blot analysis of pY118-Paxillin levels in YUMM1.7 cells overexpressing GFP-FAK in vitro and in vivo. Actin is used as a loading control. (F) Quantification of pY118-Paxillin/Paxillin levels in E. GFP control tumors are normalized to 1. n = 4 technical replicates. Error bars are mean ± SD. Non-parametric unpaired t test. (G–J) Co-immunoprecipitation analyses of CRKII and Paxillin in YUMM1.7 cell lines that exogenously express mammalian wildtype, Y118E and Y118F Paxillin in vitro (G and H) and in in vivo tumors (I and J). (H and J) Quantification of CRKII/Paxillin ratio from G and I, bands from cells expressing wildtype Paxillin are normalized to 1 both in vitro and in vivo. n = 3 technical replicates. Non-parametric one-way ANOVA, error bars are mean ± SD. (K) Coimmunoprecipitation analyses of DOCK180/RacGEF and Paxillin in YUMM1.7 cell lines that exogenously express mammalian wildtype, Y118E and Y118F Paxillin in vitro and in in vivo tumors. (L–N) Coimmunoprecipitation analyses of CRKII and DOCK180/RacGEF to Paxillin in YUMM1.7 cell lines that exogenously express wildtype Paxillin in in vitro and in in vivo tumors. (M) Quantification of CRKII/Paxillin levels in L. n = 4 tumors. (N) Quantification of DOCK180/Paxillin levels in L. n = 4 tumors. Error bars are mean ± SD. Non-parametric unpaired t test. Source data are available for this figure: .

Article Snippet: Primary antibodies used were rabbit anti-Paxillin (1:1,000, STJ94969; Antibodyplus), rabbit anti-pY118-Paxillin (1:1,000, 9369; Cell Signaling Technology), rabbit anti-FAK (1:1,000, 3285; Cell Signaling Technology), rabbit anti-pFAK397 (1:1,000, 3283; Cell Signaling Technology), chicken anti-GFP (1:500, ab13970; Abcam), mouse anti-CrkII (1:1,000610035; BD Bioscience), mouse anti-DOCK180 (1:500, sc-13163; Santa Cruz Biotechnology), mouse anti-C3G (1:250, sc-178403; Santa Cruz Biotechnology), rabbit p-ERK (1:1,000, 9101S; Cell Signaling Technology), and rabbit anti-pY31-Paxillin (1:1,000, 44-720G; Thermo Fisher Scientific).

Techniques: In Vivo, In Vitro, Cell Culture, Activation Assay, Migration, Western Blot, Expressing, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Selective PPARγ modulator diosmin improves insulin sensitivity and promotes browning of white fat

doi: 10.1016/j.jbc.2023.103059

Figure Lengend Snippet: Acute diosmin (Dios) administration improves diabetic gene programs in iWAT of mice. A , experimental model of acute control (Con) or Dios administration in mice with iWAT unilateral injection (n = 4). B , protein levels of S273 p-PPARγ, ( C ) p-IRβ, p-AKT, and p-GSK3β, ( D ) expression of gene set regulated by PPARγ S273 phosphorylation in iWAT of mice after acute Dios administration. Data are presented as mean ± SEM and ∗ p < 0.05, ∗∗ p < 0.01 compared with control group. iWAT, inguinal white adipose tissue; PPARγ, peroxisome proliferator–activated receptor γ.

Article Snippet: Membranes were incubated in 5% bovine serum albumin for 2 h and with primary antibodies overnight at 4 °C, including anti-p-PPARγ (1:2000 dilution) (catalog no.: bs-4888R; Bioss biotech), anti-PPARγ (1:1000 dilution) (catalog no.: sc-7273; Santa Cruz), anti-p-IRβ (1:2000 dilution) (catalog no.: 3025; Cell Signaling Technology), anti-p-AKT (1:2000 dilution) (catalog no.: 13038; Cell Signaling Technology), anti-p-GSK3β (1:2000 dilution) (catalog no.: 9322; Cell Signaling Technology) (1:2000 dilution), anti-UCP1 (catalog no.: Ab10983; Abcam), or β-actin (1:2000 dilution) (catalog no.: sc-47778; Santa Biotechnology).

Techniques: Injection, Expressing

Journal: Cancers

Article Title: P38 Mediates Tumor Suppression through Reduced Autophagy and Actin Cytoskeleton Changes in NRAS-Mutant Melanoma

doi: 10.3390/cancers15030877

Figure Lengend Snippet: List of primary antibodies.

Article Snippet: Phospho GSK-ß 9322 , 1:1000 , Cell Signaling , LC3 ab51520 , 1:3000 , Abcam.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: New Monoterpenoid Indole Alkaloids from Tabernaemontana crassa Inhibit β -Amyloid42 Production and Phospho-Tau (Thr217)

doi: 10.3390/ijms24021487

Figure Lengend Snippet: Results of biological activity assays. ( A ) The morphology of the U251-APP cells treated with or without compounds (5 μM or 20 μM), gemfibrozil (Gem, 50 μM, a positive control), or Dinacilib (Dina, 2.5 μM, a positive control) for 24 h. ( B ) Level of extracellular Aβ42 in the culture medium of U251-APP cells treated with compounds, Gem, or DMSO (control), determined by ELISA. ( C – E ) Levels of pTau217, pTau396 and pTau181 in the U251-APP cells treated with compounds, Dina, or DMSO (control) determined by ELISA. ( F – K ) Western blot assays showing the protein levels of CDK5, pCDK5, and GSK3 β in the U251-APP cells treated with or without compounds. A representative Western blot result ( F , H , J ) and quantification of protein levels ( G , I , K ) based on three independent experiments. ( L – Q ) Western blot assays showing the protein levels of BACE1, NCSTN, PSEN2, and PSEN1 in the U251-APP cells treated with or without compounds. A representative Western blot result ( L , N , P ) and quantification of protein levels ( M , O , Q ) based on three independent experiments. ( R ) A proposed potential role of 1 against AD by downregulating BACE1, NCSTN, CDK5, and GSK3 β -mediated pathways, resulting in A β 42 reduction and decreased pTau217. Data are presented as the means ± SD; ns, not significant; ***, p < 0.001; **, p < 0.01; and *, p < 0.05; one-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: The primary antibodies were BACE1 (Cell Signaling Technology, 5606, Danvers, MA, USA), CDK5 (Santa Cruz Biotechnology, sc-6247, Dallas, TX, USA), GSK3 β (D5C5Z) (Cell Signaling Technology, 12456), GAPDH (glyceraldehyde-3-phosphate dehydrogenase, Proteintech, 60004-1-Ig), NICSTN (Cell Signaling Technology, 5665), PSEN1 (Cell Signaling Technology, 5643), PSEN2 (Cell Signaling Technology, 9979), phospho-CDK5 (Tyr15) (pCDK5) (Absin, abs130996).

Techniques: Activity Assay, Positive Control, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Neural Regeneration Research

Article Title: Icariin ameliorates memory deficits through regulating brain insulin signaling and glucose transporters in 3×Tg-AD mice

doi: 10.4103/1673-5374.344840

Figure Lengend Snippet: Experimental scheme for this study. After gene identification at 1 month of age, 3-month-old male WT and 3×Tg-AD mice were randomly assigned to four groups with 10 animals each and then intragastrically administered either ICA or vehicle for 5 months (WT + vehicle, WT + ICA, 3×Tg-AD + vehicle, 3×Tg-AD + ICA groups). After performing behavior tests, the mice were euthanized. The cerebral cortexes were evaluated using HE and Nissl staining, immunofluorescent staining, and western blot assays to determine the above disease indicators. 3×Tg-AD: A triple-transgenic mouse model of Alzheimer’s disease; Aβ: beta-amyloid protein; AKT: protein kinase B; APP: amyloid precursor protein; GLUT: glucose transporter; GSK3β: glycogen synthase kinase 3 beta; HE: hematoxylin and eosin; ICA: icariin; IR: insulin receptor; IRS1: insulin receptor substrate 1; NeuN: neuronal nuclear antigen; p: phosphorylation; PI3K: phosphatidylinositol 3-kinase; PSD95: postsynaptic density protein 95; WT: wild-type.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-insulin (1:1000; Proteintech, Cat# 15848-1-AP, RRID: AB_10597100), rabbit anti-insulin receptor substrate 1 (IRS1; 1:1000; Proteintech Cat# 17509-1-AP, RRID: AB_10596914), mouse anti-GLUT1 (1:1000; Proteintech, Cat# 66290-1-Ig, RRID: AB_2881673), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (1:50,000; Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436), mouse anti-NeuN (1:1000, described above), rabbit anti-insulin receptor (IR) beta-subunit (1:1000; Cell Signaling Technology, Cat# 3025S, RRID: AB_2280448), rabbit anti-p-IRS1 Ser307 (1:1000; Cell Signaling Technology, Cat# 2381, RRID: AB_330342), rabbit anti-phosphatidylinositol 3-kinase (PI3K; 1:1000; Cell Signaling Technology, Cat# 4257, RRID: AB_659889), rabbit anti-phospho (p)-PI3K (1:1000; Cell Signaling Technology, Cat# 4228S, RRID: AB_659940), rabbit anti-protein kinase B (AKT; 1:1000; Cell Signaling Technology, Cat# 9272S, RRID: AB_329827), rabbit anti-glycogen synthase kinase 3 beta (GSK3β; 1:1000; Cell Signaling Technology, Cat# 9315, RRID: AB_490890), rabbit anti-p-GSK3β Ser9 (1:1000; Cell Signaling Technology, Cat# 9323, RRID: AB_2115201), rabbit anti-postsynaptic density protein 95 (PSD95; 1:1000; Abcam, Cat# ab18258, RRID: AB_444362), rabbit anti-APP (1:2000; Abcam, Cat# ab32136, RRID: AB_2289606), rabbit anti-Aβ 1–42 (1:1000; Abcam, Cat# ab201060, RRID: AB_2818982), rabbit anti-Aβ 1–40 (1:1000; Abcam, Cat# ab110888, RRID: AB_10890827), rabbit anti-PHF1 antibody (recognizing p-tau Ser396/404; 1:5000; Abcam, Cat# ab184951, RRID: AB_2861270), rabbit anti-p-tau Thr231 (1:5000; Abcam, Cat# ab151559, RRID: AB_2893278), rabbit anti-p-tau Ser199/202 (1:1000; Innovative Research, Cat# 44-768G, RRID: AB_1502103; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-p-tau Thr217 (1:1000; Innovative Research, Cat# 44-744, RRID: AB_1502121), rabbit anti-p-IR Tyr1361 (1:1000; Thermo Fisher Scientific, Cat# PA5-38283, RRID: AB_2554884), rabbit anti-p-IRS1 Ser616 (1:1000, Innovative Research, Cat# 44-550G, RRID: AB_1501245), rabbit anti-p-AKT Ser473 (1:1000; Affinity Biosciences, Zhenjiang, China, Cat# AF0016, RRID: AB_2810275), and rabbit anti-GLUT3 (1:1000; Affinity Biosciences, Cat# AF5463, RRID: AB_2837947).

Techniques: Staining, Western Blot, Transgenic Assay

Journal: Neural Regeneration Research

Article Title: Icariin ameliorates memory deficits through regulating brain insulin signaling and glucose transporters in 3×Tg-AD mice

doi: 10.4103/1673-5374.344840

Figure Lengend Snippet: Effects of ICA on impaired insulin signaling in the cerebral cortex of 3×Tg-AD mice. (A) Insulin signaling: IR tyrosine autophosphorylation is stimulated by insulin and triggers IRS1 phosphorylation at tyrosine residues, which represents a positive regulatory mechanism that activates the PI3K/AKT pathway and results in the inhibition of GSK3β. However, serine phosphorylation of IRS1 at specific sites is a negative regulatory mechanism. (B) Representative expression patterns of molecules related to the insulin signaling pathway. (C) Quantification of proteins related to the insulin signaling pathway shown in (B). Protein levels were normalized to those in the WT + vehicle group. The data are presented as the means ± SEM ( n = 4–6). * P < 0.05, vs . WT + vehicle group; # P < 0.05, vs . 3×Tg-AD + vehicle group (one-way analysis of variance followed by the least significant difference test). 3×Tg-AD: A triple-transgenic mouse model of Alzheimer’s disease; AKT: protein kinase B; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSK3β: glycogen synthase kinase 3 beta; ICA: icariin; IR: insulin receptor; IRS1: insulin receptor substrate 1; p: phosphorylation; PI3K: phosphatidylinositol 3-kinase; PIP2: phosphatidylinositol (4,5) bisphosphate; PIP3: phosphatidylinositol (3,4,5) trisphosphate; PTEN: phosphatase and tensin homolog; WT: wild-type.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-insulin (1:1000; Proteintech, Cat# 15848-1-AP, RRID: AB_10597100), rabbit anti-insulin receptor substrate 1 (IRS1; 1:1000; Proteintech Cat# 17509-1-AP, RRID: AB_10596914), mouse anti-GLUT1 (1:1000; Proteintech, Cat# 66290-1-Ig, RRID: AB_2881673), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (1:50,000; Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436), mouse anti-NeuN (1:1000, described above), rabbit anti-insulin receptor (IR) beta-subunit (1:1000; Cell Signaling Technology, Cat# 3025S, RRID: AB_2280448), rabbit anti-p-IRS1 Ser307 (1:1000; Cell Signaling Technology, Cat# 2381, RRID: AB_330342), rabbit anti-phosphatidylinositol 3-kinase (PI3K; 1:1000; Cell Signaling Technology, Cat# 4257, RRID: AB_659889), rabbit anti-phospho (p)-PI3K (1:1000; Cell Signaling Technology, Cat# 4228S, RRID: AB_659940), rabbit anti-protein kinase B (AKT; 1:1000; Cell Signaling Technology, Cat# 9272S, RRID: AB_329827), rabbit anti-glycogen synthase kinase 3 beta (GSK3β; 1:1000; Cell Signaling Technology, Cat# 9315, RRID: AB_490890), rabbit anti-p-GSK3β Ser9 (1:1000; Cell Signaling Technology, Cat# 9323, RRID: AB_2115201), rabbit anti-postsynaptic density protein 95 (PSD95; 1:1000; Abcam, Cat# ab18258, RRID: AB_444362), rabbit anti-APP (1:2000; Abcam, Cat# ab32136, RRID: AB_2289606), rabbit anti-Aβ 1–42 (1:1000; Abcam, Cat# ab201060, RRID: AB_2818982), rabbit anti-Aβ 1–40 (1:1000; Abcam, Cat# ab110888, RRID: AB_10890827), rabbit anti-PHF1 antibody (recognizing p-tau Ser396/404; 1:5000; Abcam, Cat# ab184951, RRID: AB_2861270), rabbit anti-p-tau Thr231 (1:5000; Abcam, Cat# ab151559, RRID: AB_2893278), rabbit anti-p-tau Ser199/202 (1:1000; Innovative Research, Cat# 44-768G, RRID: AB_1502103; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-p-tau Thr217 (1:1000; Innovative Research, Cat# 44-744, RRID: AB_1502121), rabbit anti-p-IR Tyr1361 (1:1000; Thermo Fisher Scientific, Cat# PA5-38283, RRID: AB_2554884), rabbit anti-p-IRS1 Ser616 (1:1000, Innovative Research, Cat# 44-550G, RRID: AB_1501245), rabbit anti-p-AKT Ser473 (1:1000; Affinity Biosciences, Zhenjiang, China, Cat# AF0016, RRID: AB_2810275), and rabbit anti-GLUT3 (1:1000; Affinity Biosciences, Cat# AF5463, RRID: AB_2837947).

Techniques: Inhibition, Expressing, Transgenic Assay

Journal: Neural Regeneration Research

Article Title: Icariin ameliorates memory deficits through regulating brain insulin signaling and glucose transporters in 3×Tg-AD mice

doi: 10.4103/1673-5374.344840

Figure Lengend Snippet: Schematic diagram of the mechanism by which ICA regulates GLUTs and brain insulin signaling to ameliorate memory impairment in AD. Aβ: Amyloid-beta protein; AD: Alzheimer’s disease; AKT: protein kinase B; APP: amyloid precursor protein; GLUT: glucose transporter; GSK3β: glycogen synthase kinase 3 beta; G-tau: the attachment of O-linked N-acetylglucosamine (O-GlcNAc) on tau; ICA: icariin; IR: insulin receptor; IRS1: insulin receptor substrate 1; p: phosphorylation; PI3K: phosphatidylinositol 3-kinase; PIP2: phosphatidylinositol (4,5) bisphosphate; PIP3: phosphatidylinositol (3,4,5) trisphosphate; PTEN: phosphatase and tensin homolog.

Article Snippet: The primary antibodies used in this study were as follows: rabbit anti-insulin (1:1000; Proteintech, Cat# 15848-1-AP, RRID: AB_10597100), rabbit anti-insulin receptor substrate 1 (IRS1; 1:1000; Proteintech Cat# 17509-1-AP, RRID: AB_10596914), mouse anti-GLUT1 (1:1000; Proteintech, Cat# 66290-1-Ig, RRID: AB_2881673), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (1:50,000; Proteintech, Cat# 60004-1-Ig, RRID: AB_2107436), mouse anti-NeuN (1:1000, described above), rabbit anti-insulin receptor (IR) beta-subunit (1:1000; Cell Signaling Technology, Cat# 3025S, RRID: AB_2280448), rabbit anti-p-IRS1 Ser307 (1:1000; Cell Signaling Technology, Cat# 2381, RRID: AB_330342), rabbit anti-phosphatidylinositol 3-kinase (PI3K; 1:1000; Cell Signaling Technology, Cat# 4257, RRID: AB_659889), rabbit anti-phospho (p)-PI3K (1:1000; Cell Signaling Technology, Cat# 4228S, RRID: AB_659940), rabbit anti-protein kinase B (AKT; 1:1000; Cell Signaling Technology, Cat# 9272S, RRID: AB_329827), rabbit anti-glycogen synthase kinase 3 beta (GSK3β; 1:1000; Cell Signaling Technology, Cat# 9315, RRID: AB_490890), rabbit anti-p-GSK3β Ser9 (1:1000; Cell Signaling Technology, Cat# 9323, RRID: AB_2115201), rabbit anti-postsynaptic density protein 95 (PSD95; 1:1000; Abcam, Cat# ab18258, RRID: AB_444362), rabbit anti-APP (1:2000; Abcam, Cat# ab32136, RRID: AB_2289606), rabbit anti-Aβ 1–42 (1:1000; Abcam, Cat# ab201060, RRID: AB_2818982), rabbit anti-Aβ 1–40 (1:1000; Abcam, Cat# ab110888, RRID: AB_10890827), rabbit anti-PHF1 antibody (recognizing p-tau Ser396/404; 1:5000; Abcam, Cat# ab184951, RRID: AB_2861270), rabbit anti-p-tau Thr231 (1:5000; Abcam, Cat# ab151559, RRID: AB_2893278), rabbit anti-p-tau Ser199/202 (1:1000; Innovative Research, Cat# 44-768G, RRID: AB_1502103; Thermo Fisher Scientific, Waltham, MA, USA), rabbit anti-p-tau Thr217 (1:1000; Innovative Research, Cat# 44-744, RRID: AB_1502121), rabbit anti-p-IR Tyr1361 (1:1000; Thermo Fisher Scientific, Cat# PA5-38283, RRID: AB_2554884), rabbit anti-p-IRS1 Ser616 (1:1000, Innovative Research, Cat# 44-550G, RRID: AB_1501245), rabbit anti-p-AKT Ser473 (1:1000; Affinity Biosciences, Zhenjiang, China, Cat# AF0016, RRID: AB_2810275), and rabbit anti-GLUT3 (1:1000; Affinity Biosciences, Cat# AF5463, RRID: AB_2837947).

Techniques:

Journal: Disease Markers

Article Title: Leptin Promotes HTR-8/SVneo Cell Invasion via the Crosstalk between MTA1/WNT and PI3K/AKT Pathways

doi: 10.1155/2022/7052176

Figure Lengend Snippet: Leptin mediates β -catenin activation through the crosstalk between MTA1/WNT and PI3K/AKT pathways in HTR-8/SVneo cells. (a) HTR-8/SVneo cells were treated with exogenous leptin (0 and 200 ng/ml) for 24 h, and MTA1, WNT1, p-GSK3 β (Ser9), and p-AKT (Ser473) levels were detected by Western blot. Data are shown as mean ± SD; ∗ P < 0.01 vs. leptin (0 ng/ml). (b) The knockdown efficiencies of MTA1 and WNT1 were analyzed by Western blot analysis and qRT-PCR. ∗ P < 0.05 vs. control. (c) HTR-8/SVneo cells were transfected with MTA1 siRNA or scramble siRNA (Scr) in the presence or absence of 200 ng/ml leptin for 24 h, and Western blot analysis was performed to detect the expression of MTA1, WNT1, p-GSK3 β (Ser9), and nuclear β -catenin. ∗ P < 0.01 vs. control and # P < 0.01 vs. leptin. (d) HTR-8/SVneo cells were transfected with WNT1 siRNA or scramble siRNA (Scr) in the presence or absence of 200 ng/ml leptin for 24 h, and Western blot analysis was performed to detect the expression of WNT1, p-GSK3 β (Ser9), and nuclear β -catenin. ∗ P < 0.01 vs. control and # P < 0.01 vs. leptin. (e) The knockdown efficiencies of AKT and β -catenin were analyzed by Western blot analysis and qRT-PCR. ∗ P < 0.05 vs. control. (f) HTR-8/SVneo cells were transfected with AKT siRNA or scramble siRNA (Scr) in the presence or absence of 200 ng/ml leptin for 24 h, and Western blot analysis was performed to detect the expression of AKT, p-GSK3 β (Ser9), and nuclear β -catenin. ∗ P < 0.01 vs. control and # P < 0.01 vs. leptin. All experiments were performed in triplicate.

Article Snippet: The following primary antibodies were used: MMP9 (ab76003), MTA1 (ab71153), WNT1 (ab15251), AKT (ab179463), GSK3 β (ab32391), β -catenin (ab32572), histone 3 (ab1791), β -actin (ab6276) (Abcam, USA); p-AKT (Ser473) (#4060), p-GSK3 β (Ser9) (#9322) (Cell Signaling Technology, USA).

Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Transfection, Expressing

Journal: Respiratory Research

Article Title: Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: I. Effects on lung remodeling and pathology

doi: 10.1186/1465-9921-14-113

Figure Lengend Snippet: Effect of repeated intranasal LPS challenge and treatment with the selective GSK-3 inhibitor SB216763 on extracellular matrix deposition in the lung. (A) Expression of fibronectin was evaluated in whole lung homogenates 24 hours after the last challenge by immunoblotting using specific antibodies. Equal protein loading was verified by the analysis of GAPDH. Effects of repeated LPS challenge and SB216763 treatment on fibronectin expression were quantified by densitometry, representing mean ± s.e.m. of 9 animals per group. (B) Histological staining of the extracellular matrix protein collagen using Sirius Red. The non-cartilaginous airways were digitally photographed (100-200 × magnification) and analysed by using ImageJ software. Effects of repeated LPS challenge and SB216763 treatment on airway collagen expression were quantified, representing mean ± s.e.m. of 9 animals per group. (C) The mean linear intercept (LMI), a measure for alveolar airspace size, was determined by staining the tissue-sections with haematoxylin and eosin. Data represent means ± s.e.m. of 9 animals per group. **p < 0.01 compared to control group and # p < 0.05 compared to LPS treated animals. Scale bar = 200 μm.

Article Snippet: Rabbit anti-phospho-Ser9/21-GSK-3 antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Western Blot, Staining, Software

Journal: Respiratory Research

Article Title: Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: I. Effects on lung remodeling and pathology

doi: 10.1186/1465-9921-14-113

Figure Lengend Snippet: Repeated LPS instillation and pharmacological inhibition of GSK-3 by SB216763 do not affect airway smooth muscle content. Immunohistological analysis of sm-MHC positive area in (A) large (cartilaginous) and (B) small (non-cartilaginous) airways. Effects of repeated LPS challenge and SB216763 treatment on airway smooth muscle sm-MHC expression were quantified. Data represent means ± s.e.m. of 9 animals per group. Scale bar = 200 μm.

Article Snippet: Rabbit anti-phospho-Ser9/21-GSK-3 antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Inhibition, Expressing

Journal: Respiratory Research

Article Title: Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: I. Effects on lung remodeling and pathology

doi: 10.1186/1465-9921-14-113

Figure Lengend Snippet: Right ventricle hypertrophy after repeated intranasal LPS instillation is attenuated by GSK-3 inhibition. Effect of repeated LPS instillation and GSK-3 inhibition by SB216763 on right ventricle hypertrophy. Effects of repeated LPS challenge and SB216763 treatment on size of right ventricle were quantified as right ventricle weight over total heart weight, representing mean ± s.e.m. of 9 animals per group. **p < 0.01 compared to control group and # p < 0.05 compared to LPS treated animals.

Article Snippet: Rabbit anti-phospho-Ser9/21-GSK-3 antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Inhibition

Journal: Respiratory Research

Article Title: Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: I. Effects on lung remodeling and pathology

doi: 10.1186/1465-9921-14-113

Figure Lengend Snippet: GSK-3 inhibition does not inhibit LPS-induced pulmonary inflammation. Effect of repeated LPS instillation and GSK-3 inhibition by SB216763 on inflammatory cell infiltration in the airways. Cells within 50 μm of the basement membrane were quantified and expressed relative to basement membrane length, representing mean ± s.e.m. of 9 animals per group. *p < 0.05 compared to control group. Scale bar = 200 μm.

Article Snippet: Rabbit anti-phospho-Ser9/21-GSK-3 antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Inhibition

Journal: Respiratory Research

Article Title: Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: I. Effects on lung remodeling and pathology

doi: 10.1186/1465-9921-14-113

Figure Lengend Snippet: Activation of β-catenin in response to repeated intranasal LPS challenge is prevented by treatment with the selective GSK-3 inhibitor SB216763. (A) Expression of active β-catenin, phosphorylated GSK-3 (ser9/21 GSK-3) and total GSK-3 was evaluated in whole lung homogenates 24 hours after the last challenge by immunoblotting using specific antibodies. Equal protein loading was verified by the analysis of GAPDH. (B,C) Responses of repeated LPS challenge and SB216763 treatment on active β-catenin expression (B) and GSK-3 phosphorylation (C) were quantified by densitometry, representing mean ± s.e.m. of 9 animals per group. (D) Correlation between pulmonary expression of fibronectin (data from Figure ) and active β-catenin in all guinea pigs. R = 0.552; p < 0.001. (E) Immunofluorescence analysis of active β-catenin (red) in large airways counterstained with Hoechst 3342 to stain nuclei (blue). *p < 0.05 compared to control group and # p < 0.05 compared to LPS treated animals.

Article Snippet: Rabbit anti-phospho-Ser9/21-GSK-3 antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Expressing, Western Blot, Immunofluorescence, Staining