human phosphokinase array  (Cell Signaling Technology Inc)

Journal: MedComm

Article Title: Prolyl 4‐hydroxylase α‐subunit family regulation of type I collagen deposition and IL17RB/c‐Jun activation synergistically mediate choline dehydrogenase promotion of colorectal cancer metastasis

doi: 10.1002/mco2.70007

Figure Lengend Snippet: Choline dehydrogenase (CHDH) mediates c‐Jun activation to promote colorectal cancer (CRC) cell migration in vitro. (A, B) Activation of 39 kinases or key proteins were detected in RKO cells overexpressing CHDH or MCS using a human phosphokinase array. (C) Western blot analysis the expression of c‐Jun and p‐c‐Jun in CHDH knockdown or overexpression cells. (D, E) Representative immunohistochemistry (IHC) staining of CHDH and p‐c‐Jun in human CRC tissues and the correlation results were shown, scale bar: 50 µm. (F) Western blot analysis the expression of c‐Jun and p‐c‐Jun in CHDH overexpression cells treated with SP600125. (G) Wound‐healing assay images of RKO and LOVO expressing CHDH cells treated with DMSO (Ctrl) or SP600125 were shown. Scale bar: 50 µm. (H) Transwell assay images of RKO and LOVO expressing CHDH cells treated with DMSO (Ctrl) or SP600125 were shown. Scale bar: 50 µm. (I) Statistic results on wound closure in RKO and LOVO expressing CHDH cells treated with DMSO (Ctrl) or SP600125. ** p < 0.01, *** p < 0.001. (J) Statistic results on migrated cells in RKO and LOVO expressing CHDH cells treated with DMSO (Ctrl) or SP600125. *** p < 0.001. (K) Immunofluorescence assay to detect F‐actin rearrangement in RKO and LOVO expressing CHDH cells treated with DMSO (Ctrl) or SP600125 via phalloidin staining. Scale bar: 20 µm. (L) Western blot analysis the expression of epithelial–mesenchymal transition (EMT) markers in RKO and LOVO expressing CHDH cells treated with DMSO (Ctrl) or SP600125.

Article Snippet: The Human Phosphokinase Array Kit was purchased from R&D Systems (Cat. #ARY003C), then was used to quantify relative kinase and protein phosphorylation levels following the manufacturer's instructions.,

Techniques: Activation Assay, Migration, In Vitro, Western Blot, Expressing, Knockdown, Over Expression, Immunohistochemistry, Wound Healing Assay, Transwell Assay, Immunofluorescence, Staining

Journal: Nature Cardiovascular Research

Article Title: Network-based prioritization and validation of regulators of vascular smooth muscle cell proliferation in disease

doi: 10.1038/s44161-024-00474-4

Figure Lengend Snippet: a , Phosphokinase array and densitometric quantification of serum-starved hVSMCs treated for 15 min with 500 ng ml −1 rhTIMP1 or vehicle control ( N = 1 donor). b , Western blot of total STAT3, phospho-STAT3 (S727 or Tyr705), total AKT, phospho-AKT (pAKT), total p38a, phospho-p38a (p-p38a) and GAPDH in serum-starved hVSMCs after 0 min, 5 min, 10 min, 15 min and 30 min rhTIMP1. c , Quantification of relative western blot band intensity, normalized to GAPDH. The points show independent hVSMC isolates ( N = 4), the bars indicate the means, and the error bars indicate s.e.m.; P value: one-way ANOVA. d , Number of clonally expanded patches formed by lineage-labeled VSMCs following 21 days of culture, without (white, circles) or with 500 ng rmTIMP1 (blue, squares) in samples treated with vehicle (DMSO), 10 µM TT101 (STAT3i), 100 nM MK-2206 (AKTi), 10 µM SB202474 (control inhibitor, ctrl), 10 µM SB203580 (p38i1) or 10 µM SB202190 (p38i2). The points show averages ( N = 3 Myh11–Confetti animals analyzed in triplicate), the bars indicate means, the error bars indicated s.e.m.; P value: two-tailed t -test. e , Quantification over time of clonally expanded patches formed by lineage-labeled VSMCs, treated with non-targeting control (NTC) or Stat3 -targeting siRNA (siSTAT3) ±500 ng rmTIMP1. The points indicate means ( N = 3 Myh11–Confetti animals analyzed in triplicate), and the error bars indicated s.e.m.; P = 2.2 × 10 −7 , generalized linear model. f , ChIP–qPCR analysis at STAT3 targets ( TWIST and JUNB ) and negative control ( AMICA1 ), in serum-starved control and rhTIMP1-treated hVSMCs (15 min, 500 ng ml −1 ), showing anti-STAT3 and control-IgG precipitated DNA as a percentage of the input. The bars show the means of independent hVSMC isolates ( N = 3 donors), and the error bars indicate the s.e.m.; P value: two-tailed t -test. g , pSTAT3 S727 immunostaining (magenta) in cryosections from carotid plaque in an Myh11–Confetti/Apoe animal (11 weeks HFD) with Myh11–Confetti signals (CFP, blue; RFP, red; YFP, yellow; GFP, green). Representative of three animals. Scale bar = 50 µm (applies to both images in panel). h , i , pSTAT3 S727 and KI67 immunostaining ( h ) and quantification ( i ) of serum-starved control hVSMCs ±500 ng ml −1 rhTIMP1 treatment (15 min, h ) or indicated time points ( i ). The arrowheads indicate KI67 + cells. Scale bars = 50 µm. The symbols show average values for independent hVSMC isolates ( N = 3 donors, analyzed in triplicate), the lines represent means and the error bars represent s.e.m.; P value: two-way ANOVA, * P = 0.0498; NS, not significant.

Article Snippet: Phosphokinase array (R&D Systems, ARY003C) was incubated with cell lysates according to the manufacturer’s instructions.

Techniques: Control, Western Blot, Labeling, Two Tailed Test, Negative Control, Immunostaining

Journal: Nature Cardiovascular Research

Article Title: Network-based prioritization and validation of regulators of vascular smooth muscle cell proliferation in disease

doi: 10.1038/s44161-024-00474-4

Figure Lengend Snippet: a , Heatmap showing relative spot intensity of phosphokinase array analysis of hVSMCs following 15 minutes 500 ng/mL rhTIMP1 or vehicle control treatment on a black (low) to red (high) scale [AU]. N = 1. b , Western blots of hVSMCs from 4 different donors showing total, pS727 and pTyr705 STAT3, total and phospho AKT, total and phospho-p38 (p-p38), and GAPDH in serum-starved cells after 0, 5 10, 15 or 30 minutes rhTIMP1 treatment. c , Quantification of relative band intensity in ( b ) for total STAT3, total AKT and total p38 protein levels, normalized to GAPDH. Points show independent hVSMC isolates (N = 4 donors), bars mean, error bars SEM. d , pSTAT3 S727 immunostaining (magenta) in cryosections from injured carotid artery of Myh11-Confetti (top, N = 3 animals) or atherosclerotic plaque from Myh11-Confetti/Apoe animals fed a HFD (lower panel, N = 3). Signals for nuclear DAPI (white) and confetti lineage-label are also shown. White box indicate region shown in Fig. . Scale bar= 50 μm. e , %KI67+ hVSMCs in samples treated with 500 ng/mL rhTIMP1 following serum-starvation for indicated timepoint. Symbols show average values for independent hVSMC isolates (N = 3, analyzed in triplicate), lines the mean, error bars SEM. f , Nuclear pSTAT3 (S727) intensity in FACS-isolated EYFP+ mVSMCs without (CTRL, dots) or with 500 ng/mL rmTIMP1 (squares) either 4 (left) or 7 days post seeding (right). Symbols show average values from independent (N = 4 Myh11-EYFP animals analyzed in quadruplicate), lines mean, error bars SEM, p-value: two-tailed t -test.

Article Snippet: Phosphokinase array (R&D Systems, ARY003C) was incubated with cell lysates according to the manufacturer’s instructions.

Techniques: Control, Western Blot, Immunostaining, Isolation, Two Tailed Test