Journal: bioRxiv

Article Title: Tip growth in the brown alga Ectocarpus is controlled by a RHO-GAP-BAR domain protein independently from F-actin organisation

doi: 10.1101/2021.08.28.458042

Figure Lengend Snippet: For each cell both bright field and epifluorescence or confocal microscopy are shown. A . Negative control for the immunolocalisation of G-actin. Only the secondary antibody was added. B - E . Immunolocalisation of G-actin with a rabbit anti-plant actin polyclonal antibody. F - M . Alexa-488-Phalloidin labelling. B , C , F , G , H , M . WT. D , E , I , J , K , L : etl . M . treated with 3µM latrunculin B in 0.1% DMSO. A - G , M : epifluoresence microscopy, H - L : confocal microscopy. Scale bar: 50µm in A , 10µm for the other photos.

Article Snippet: After rinsing twice 10min with 1 % BSA in PBS, 100 µL of polyclonal antibodies ETOILE-BAR and ETOILE-RHOGAP (1:500), or of the rabbit anti-Human Rac1 polyclonal antibodies (1:40, Thermofisher PA1-091) were added in 1 % BSA-PBS and incubated O/N at 4°C.

Techniques: Confocal Microscopy, Negative Control, Microscopy

Journal: BMC Cancer

Article Title: High MICAL1 expression correlates with cancer progression and immune infiltration in renal clear cell carcinoma

doi: 10.1186/s12885-022-10462-1

Figure Lengend Snippet: The effect of MICAL1 on Rac1 activation and renal clear cell carcinoma cell migration. a Caki-1 and 786-O cells were transfected with control siRNA or siRNA specifically targeting MICAL1 (siMICAL1) for 48 h, then total protein extracts from those cells were analyzed for MICAL1 expression. Western blot bands corresponding to MICAL1 were quantified and normalized against GAPDH. The statistical graph from Caki-1 cells was shown in Fig. panel on the right. b After knockdown of MICAL1 for 48 h, the proliferation rates of Caki-1 and 786-O cells were assessed by CCK8 assays. (C&D) After knockdown of MICAL1 for 36 h, the migratory rates of these cells were assessed by Transwell c and wound healing assays d . e Levels of GTP-Rac1 in MICAL1-knockdown Caki-1 cells were assessed. * p < 0.05, ** p < 0.01

Article Snippet: Antibodies targeting the following proteins were used: the polyclonal rabbit anti-human MICAL1 antibody (14818–1-AP, Proteintech, Wuhan, China), the polyclonal rabbit anti-human Rac1 antibody (24072–1-AP, Proteintech, Wuhan, China), the polyclonal rabbit anti-human GAPDH antibody (BS72410, Bioworld, Nanjing, China), the polyclonal goat anti-rabbit IgG (H + L) HRP (ZF0101, Zfanti, Nanjing, China).

Techniques: Activation Assay, Migration, Transfection, Control, Expressing, Western Blot, Knockdown

Journal: Frontiers in Oncology

Article Title: Circulatory Level of Inflammatory Cytoskeleton Signaling Regime Proteins in Cancer Invasion and Metastasis

doi: 10.3389/fonc.2022.851807

Figure Lengend Snippet: Scatter graph illustrated level of proteins (A) . LOX5, (B) . Rac1, (C) . Rac1b, (D) . p38a, (E) . Phospho-p38a (F) LIMK1 (G) Phospho-LIMK1 (H) Cofilin1 (I) Phospho-Cofilin1 in the serum of HC, NM, M, and MF (MF-metastatic post-therapy follow-up). For each group, standard error of the mean (mean ± SE) was plotted. ANOVA test was used to compare the means between the groups *- p <0.01, **- p <0.001, ***- p <0.0001.

Article Snippet: Antibody against all the above-mentioned proteins, i.e., Rabbit anti-human Rac1 IgG, Mouse anti-human p38α IgG, Mouse anti-human phospho-p38α (Y-182) IgG, (Santa Cruz Biotechnology, CA, USA), Rabbit anti-human LOX5 IgG (Cell Signaling Technology, Danvers, Massachusetts, USA), Rabbit anti-human Rac1b IgG (Merck Millipore, Burlington, Massachusetts, USA), Rabbit anti-human LIMK1 IgG, Rabbit anti-human phospho-LIMK1 (T-508) IgG, Rabbit anti-human cofilin1 IgG, and Rabbit anti-human phospho-cofilin (S-3) IgG (St. Louis, Immunotag USA) were immobilized on a separate flow cell of CM5 sensor chip by using amine coupling kit.

Techniques:

Journal: Frontiers in Oncology

Article Title: Circulatory Level of Inflammatory Cytoskeleton Signaling Regime Proteins in Cancer Invasion and Metastasis

doi: 10.3389/fonc.2022.851807

Figure Lengend Snippet: Concentration of proteins in Healthy Control (HC), Non-metastatic (NM), Metastatic (M), and CR+PR of M group (pre- and post-therapy).

Article Snippet: Antibody against all the above-mentioned proteins, i.e., Rabbit anti-human Rac1 IgG, Mouse anti-human p38α IgG, Mouse anti-human phospho-p38α (Y-182) IgG, (Santa Cruz Biotechnology, CA, USA), Rabbit anti-human LOX5 IgG (Cell Signaling Technology, Danvers, Massachusetts, USA), Rabbit anti-human Rac1b IgG (Merck Millipore, Burlington, Massachusetts, USA), Rabbit anti-human LIMK1 IgG, Rabbit anti-human phospho-LIMK1 (T-508) IgG, Rabbit anti-human cofilin1 IgG, and Rabbit anti-human phospho-cofilin (S-3) IgG (St. Louis, Immunotag USA) were immobilized on a separate flow cell of CM5 sensor chip by using amine coupling kit.

Techniques: Concentration Assay

Journal: Frontiers in Oncology

Article Title: Circulatory Level of Inflammatory Cytoskeleton Signaling Regime Proteins in Cancer Invasion and Metastasis

doi: 10.3389/fonc.2022.851807

Figure Lengend Snippet: ROC of serum proteins between HC and BC. (A) LOX5; AUC: 0.802, (B) Rac1; AUC: 0.774, (C) Rac1b; AUC: 0.501, (D) p38α; AUC: 0.890, (E) phospho-p38α (Y-182); AUC: 0.932, (F) LIMK1, AUC: 0.915 (G) phospho-LIMK1 (T-508); AUC: 0.910, (H) Cofilin1; AUC: 0.810, (I) phospho-Cofilin1 (S-3); AUC: 0.772.

Article Snippet: Antibody against all the above-mentioned proteins, i.e., Rabbit anti-human Rac1 IgG, Mouse anti-human p38α IgG, Mouse anti-human phospho-p38α (Y-182) IgG, (Santa Cruz Biotechnology, CA, USA), Rabbit anti-human LOX5 IgG (Cell Signaling Technology, Danvers, Massachusetts, USA), Rabbit anti-human Rac1b IgG (Merck Millipore, Burlington, Massachusetts, USA), Rabbit anti-human LIMK1 IgG, Rabbit anti-human phospho-LIMK1 (T-508) IgG, Rabbit anti-human cofilin1 IgG, and Rabbit anti-human phospho-cofilin (S-3) IgG (St. Louis, Immunotag USA) were immobilized on a separate flow cell of CM5 sensor chip by using amine coupling kit.

Techniques:

Journal: Frontiers in Oncology

Article Title: Circulatory Level of Inflammatory Cytoskeleton Signaling Regime Proteins in Cancer Invasion and Metastasis

doi: 10.3389/fonc.2022.851807

Figure Lengend Snippet: ROC of serum proteins between NM and M. (A) LOX5; AUC: 0.774, (B) Rac1; AUC: 0.781, (C) Rac1b; AUC: 0.757, (D) p38α; AUC: 0.812, (E) phospho-p38α (Y-182); AUC: 0.820, (F) LIMK1, AUC: 0.807 (G) phospho-LIMK1 (T-508); AUC: 0.817, (H) Cofilin1; AUC: 0.819, (I) phospho-Cofilin1 (S-3); AUC: 0.807.

Article Snippet: Antibody against all the above-mentioned proteins, i.e., Rabbit anti-human Rac1 IgG, Mouse anti-human p38α IgG, Mouse anti-human phospho-p38α (Y-182) IgG, (Santa Cruz Biotechnology, CA, USA), Rabbit anti-human LOX5 IgG (Cell Signaling Technology, Danvers, Massachusetts, USA), Rabbit anti-human Rac1b IgG (Merck Millipore, Burlington, Massachusetts, USA), Rabbit anti-human LIMK1 IgG, Rabbit anti-human phospho-LIMK1 (T-508) IgG, Rabbit anti-human cofilin1 IgG, and Rabbit anti-human phospho-cofilin (S-3) IgG (St. Louis, Immunotag USA) were immobilized on a separate flow cell of CM5 sensor chip by using amine coupling kit.

Techniques:

Journal: Frontiers in Oncology

Article Title: Circulatory Level of Inflammatory Cytoskeleton Signaling Regime Proteins in Cancer Invasion and Metastasis

doi: 10.3389/fonc.2022.851807

Figure Lengend Snippet: Combined ROC between NM and M breast cancer patients with panel of proteins m1(phospho-LIMK1, phospho-p38α, and p38α), m2 (LOX5, Rac1, and phospho-p38α), m3 (LOX5, phospho-p38α, and phospho-cofilin1). Among these combination m1 showing excellent AUC value of 0.95, specificity 94.5%, sensitivity 89.33%, PPV 89.6%, and NPV 94.4%.

Article Snippet: Antibody against all the above-mentioned proteins, i.e., Rabbit anti-human Rac1 IgG, Mouse anti-human p38α IgG, Mouse anti-human phospho-p38α (Y-182) IgG, (Santa Cruz Biotechnology, CA, USA), Rabbit anti-human LOX5 IgG (Cell Signaling Technology, Danvers, Massachusetts, USA), Rabbit anti-human Rac1b IgG (Merck Millipore, Burlington, Massachusetts, USA), Rabbit anti-human LIMK1 IgG, Rabbit anti-human phospho-LIMK1 (T-508) IgG, Rabbit anti-human cofilin1 IgG, and Rabbit anti-human phospho-cofilin (S-3) IgG (St. Louis, Immunotag USA) were immobilized on a separate flow cell of CM5 sensor chip by using amine coupling kit.

Techniques:

Journal: Frontiers in Oncology

Article Title: Circulatory Level of Inflammatory Cytoskeleton Signaling Regime Proteins in Cancer Invasion and Metastasis

doi: 10.3389/fonc.2022.851807

Figure Lengend Snippet: Western blot image and their densitometry analysis of proteins (A) LOX5, (B) Rac1, (C) Rac1b, (D) p38α, (E) phospho-p38α (Y-182), (F) LIMK1, (G) phospho-LIMK1 (T-508), (H) Cofilin1, (I) phospho-Cofilin1 (S-3) between HC, NM, M, and MF (Lanes-1&2-HC, 3&4-NM, 5&6-M, and 7&8-MF). An unpaired t-test was used to compare the means between two groups (**p<0.01 and *p<0.05).

Article Snippet: Antibody against all the above-mentioned proteins, i.e., Rabbit anti-human Rac1 IgG, Mouse anti-human p38α IgG, Mouse anti-human phospho-p38α (Y-182) IgG, (Santa Cruz Biotechnology, CA, USA), Rabbit anti-human LOX5 IgG (Cell Signaling Technology, Danvers, Massachusetts, USA), Rabbit anti-human Rac1b IgG (Merck Millipore, Burlington, Massachusetts, USA), Rabbit anti-human LIMK1 IgG, Rabbit anti-human phospho-LIMK1 (T-508) IgG, Rabbit anti-human cofilin1 IgG, and Rabbit anti-human phospho-cofilin (S-3) IgG (St. Louis, Immunotag USA) were immobilized on a separate flow cell of CM5 sensor chip by using amine coupling kit.

Techniques: Western Blot

Journal: Apoptosis

Article Title: Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

doi: 10.1007/s10495-019-01576-2

Figure Lengend Snippet: Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p < 0.05 vs. the untreated samples)

Article Snippet: Total and phospho-Rac1 were detected using a polyclonal rabbit anti-human Rac1/2/3 antibody diluted 1:3000 (v/v) in TBST, and a polyclonal rabbit anti-human phospho-Ser71-Rac antibody diluted 1:3000 (v/v) in TBST (Cell Signalling Technologies).

Techniques: Phospho-proteomics, Western Blot, Transfection, Incubation, Expressing, Recombinant, SDS Page

Journal: Apoptosis

Article Title: Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

doi: 10.1007/s10495-019-01576-2

Figure Lengend Snippet: Analysis of Src phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP, b pCMV6-Ac-TF Ala253 -tGFP, c pCMV6-Ac-TF-tGFP plasmids, along with d an untransfected set of cells. The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed in Laemmeli’s buffer containing a protease inhibitor cocktail and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Src1 antibody, diluted 1:3000 (v/v) or a rabbit monoclonal anti-human phospho-Tyr416-Src family antibody (D49G4), diluted 1:4000 (v/v) in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Src were calculated using the ImageJ program. (n = 6; * = p < 0.05 vs. the untreated samples)

Article Snippet: Total and phospho-Rac1 were detected using a polyclonal rabbit anti-human Rac1/2/3 antibody diluted 1:3000 (v/v) in TBST, and a polyclonal rabbit anti-human phospho-Ser71-Rac antibody diluted 1:3000 (v/v) in TBST (Cell Signalling Technologies).

Techniques: Western Blot, Transfection, Incubation, Expressing, Recombinant, Protease Inhibitor, SDS Page

Journal: Apoptosis

Article Title: Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

doi: 10.1007/s10495-019-01576-2

Figure Lengend Snippet: Analysis of Rac phosphorylation by western blot. Human coronary artery endothelial cells (2 × 10 5 ) were seeded out into 12-well plates and transfected with 0.5 µg of a pCMV6-Ac-tGFP or b pCMV6-Ac-TF Ala253 -tGFP (additional samples shown in Supplementary Fig. 2). The cells were incubated for 48 h to permit the expression of the recombinant proteins. Sets of cells were then activated with PAR2-AP (20 µM) and incubated for up to 120 min. The cells were then lysed and separated by 12% (w/v) SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were probed with a polyclonal rabbit anti-human Rac1/2/3 antibody, diluted 1:3000 (v/v) and a rabbit monoclonal anti-human phospho-Ser71-Rac1 antibody, diluted 1:3000 (v/v) diluted in TBST. The membranes were then washed and probed with a goat anti-rabbit alkaline phosphatase-conjugated antibody diluted 1:1000 (v/v) and then visualised using the Western Blue stabilised alkaline phosphatase-substrate and recorded. All quantifications were normalised against GAPDH which was detected using a polyclonal goat anti-GAPDH antibody diluted 1:5000 (v/v) and then detected using an alkaline phosphatase-conjugated donkey anti-goat-IgG antibody diluted 1:2000 (v/v). The ratios of phosphorylated to total Rac were calculated using the ImageJ program. (n = 6; * = p < 0.05 vs. the untreated samples)

Article Snippet: Total and phospho-Rac1 were detected using a polyclonal rabbit anti-human Rac1/2/3 antibody diluted 1:3000 (v/v) in TBST, and a polyclonal rabbit anti-human phospho-Ser71-Rac antibody diluted 1:3000 (v/v) in TBST (Cell Signalling Technologies).

Techniques: Western Blot, Transfection, Incubation, Expressing, Recombinant, SDS Page