Journal: PLOS ONE

Article Title: VEGF-A165 is the predominant VEGF-A isoform in platelets, while VEGF-A121 is abundant in serum and plasma from healthy individuals

doi: 10.1371/journal.pone.0284131

Figure Lengend Snippet: Mixture of rhVEGF-A121, rhVEGF-A165 and rhVEGF-A189 (50 μg each) were subjected to 15% SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE). The transferred membrane was detected with anti-VEGF-A polyclonal antibody (left panel), anti-VEGF-A165 monoclonal antibody (middle panel), and anti-VEGF-A121 monoclonal antibody (right panel). The upper band of VEGF-A121 bands in SDS-PAGE (Lane 3) is due to glycosylation according to manufacturer’s instruction provided from Cell Signaling Technologies.

Article Snippet: Recombinant human VEGF-A121 (rhVEGF-A121) and VEGF-A165 (rhVEGF-A165) were purchased from Cell signaling Technologies (Danvers, MA).

Techniques: Polyacrylamide Gel Electrophoresis, SDS Page

Journal: PLOS ONE

Article Title: VEGF-A165 is the predominant VEGF-A isoform in platelets, while VEGF-A121 is abundant in serum and plasma from healthy individuals

doi: 10.1371/journal.pone.0284131

Figure Lengend Snippet: Specific immunoreactivity of the anti-VEGF-A121 and anti-VEGF-A165 monoclonal antibodies using for ELISA. The absorbance values at 450 nm (Abs 450 nm) obtained by VEGF-A121 ELISA (A, C) and VEGF-A165 ELISA (B, D). For these ELISAs, different doses of rhVEGF-A121, rhVEGF-A165, and rhVEGF-A189 (A, B) or rhVEGF-A206 (C, D) were evaluated. VEGF-A121 ELISA and VEGF-A165 ELISA specifically recognized rhVEGF-A121 (A, C) and rhVEGF-A165 (B, D), respectively.

Article Snippet: Recombinant human VEGF-A121 (rhVEGF-A121) and VEGF-A165 (rhVEGF-A165) were purchased from Cell signaling Technologies (Danvers, MA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLOS ONE

Article Title: VEGF-A165 is the predominant VEGF-A isoform in platelets, while VEGF-A121 is abundant in serum and plasma from healthy individuals

doi: 10.1371/journal.pone.0284131

Figure Lengend Snippet: (A, B) A standard curve of rhVEGF-A121 by VEGF-A121 ELISA (A) and a standard curve of rhVEGF-A165 by VEGF-A165 ELISA (B) were independently created. (C, D) Different lower detection limits for rhVEGF-A121 and rhVEGF-A165 were determined in each ELISA (n = 5); the ELISA lower detection limits for VEGF-A121 or VEGF-A165 were 35 pg/mL (C) or 38 pg/mL (D), respectively. Abs 450 nm represents absorbance at 450 nm.

Article Snippet: Recombinant human VEGF-A121 (rhVEGF-A121) and VEGF-A165 (rhVEGF-A165) were purchased from Cell signaling Technologies (Danvers, MA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLOS ONE

Article Title: VEGF-A165 is the predominant VEGF-A isoform in platelets, while VEGF-A121 is abundant in serum and plasma from healthy individuals

doi: 10.1371/journal.pone.0284131

Figure Lengend Snippet: (A) HEK293 cells were transfected with plasmids overexpressed VEGF-A121 (pVEGF-A121) and VEGF-A165 (pVEGF-A165) and empty plasmid (mock). Their cell lysates were blotted by VEGF-A polyclonal antibody and β-actin. (B—D) Total VEGF-A in the media from (A) was measured by Human VEGF Quantikine ELISA Kit (B), VEGF-A121 ELISA (C), and VEGF-A165 ELISA (D). Graph represents mean ± SD; n = 9.

Article Snippet: Recombinant human VEGF-A121 (rhVEGF-A121) and VEGF-A165 (rhVEGF-A165) were purchased from Cell signaling Technologies (Danvers, MA).

Techniques: Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: PLOS ONE

Article Title: VEGF-A165 is the predominant VEGF-A isoform in platelets, while VEGF-A121 is abundant in serum and plasma from healthy individuals

doi: 10.1371/journal.pone.0284131

Figure Lengend Snippet: Concentrations of total VEGF-A, VEGF-A121, and VEGF-A165 were measured in serum and plasma from healthy volunteers (n = 20). Correlation between total VEGF-A and VEGF-A121 or VEGF-A165 in serum (A) and plasma (B) were evaluated by a linear regression analysis (left panels of A and B: total VEGF-A vs. VEGF-A121, right panels of A and B: total VEGF-A vs. VEGF-A165). Correlations were determined using Spearman’s correlation test (two-tailed). Results are represented as significant at p < 0.05.

Article Snippet: Recombinant human VEGF-A121 (rhVEGF-A121) and VEGF-A165 (rhVEGF-A165) were purchased from Cell signaling Technologies (Danvers, MA).

Techniques: Two Tailed Test

Journal: PLOS ONE

Article Title: VEGF-A165 is the predominant VEGF-A isoform in platelets, while VEGF-A121 is abundant in serum and plasma from healthy individuals

doi: 10.1371/journal.pone.0284131

Figure Lengend Snippet: (A, B) VEGF-A121 and VEGF-A165 in plasma and serum (A) and those in platelet lysate (PL-VEGF-A121 and PL-VEGF-A165) (B) were measured by isoform specific ELISAs (n = 59). (C) VEGF-A121 released from aggregating platelets (PR-VEGF-A121) and VEGF-A121 in platelet lysate (PL-VEGF-A121) were the same levels for 10 6 number of platelets (left panel). VEGF-A165 released from aggregating platelets (PR-VEGF-A165) was significantly lower than VEGF-A165 in platelet lysate (PL-VEGF-A165) (right panel) (n = 8). (D) The ratio of PR-VEGF-A121 to PL-VEGF-A121 and that of PR-VEGF-A165 to PL-VEGF-A165 in the same number of platelets were calculated from (C). *P < 0.0001, **P < 0.05, and ***P < 0.001.

Article Snippet: Recombinant human VEGF-A121 (rhVEGF-A121) and VEGF-A165 (rhVEGF-A165) were purchased from Cell signaling Technologies (Danvers, MA).

Techniques:

Journal: BMC Ophthalmology

Article Title: Predictive effect of TCED-HFV grading and imaging biomarkers on anti-VEGF therapy in diabetic macular edema

doi: 10.1186/s12886-023-02973-7

Figure Lengend Snippet: Univariate analysis for predictors of 6-month outcomes in diabetic macular edema treated with anti-VEGF

Article Snippet: Although severe DME staging was an important factor in univariate analysis, it was not included in multivariate analysis because it was composed of other factors included in the model. Fig. 2 Multivariate analysis of imaging biomarkers and outcomes after anti-VEGF therapy in patients with diabetic macular edema (DME). (A) Endpoint 1: CST reduction of 10% or more from baseline. (B) Endpoint 2: CST < 300 μm at 6 months. (C) Endpoint 3: BCVA improvement of five letters or more from baseline.

Techniques:

Journal: BMC Ophthalmology

Article Title: Predictive effect of TCED-HFV grading and imaging biomarkers on anti-VEGF therapy in diabetic macular edema

doi: 10.1186/s12886-023-02973-7

Figure Lengend Snippet: Multivariate analysis of imaging biomarkers and outcomes after anti-VEGF therapy in patients with diabetic macular edema (DME). (A) Endpoint 1: CST reduction of 10% or more from baseline. (B) Endpoint 2: CST < 300 μm at 6 months. (C) Endpoint 3: BCVA improvement of five letters or more from baseline. * P <0.05; * P <0.01.

Article Snippet: Although severe DME staging was an important factor in univariate analysis, it was not included in multivariate analysis because it was composed of other factors included in the model. Fig. 2 Multivariate analysis of imaging biomarkers and outcomes after anti-VEGF therapy in patients with diabetic macular edema (DME). (A) Endpoint 1: CST reduction of 10% or more from baseline. (B) Endpoint 2: CST < 300 μm at 6 months. (C) Endpoint 3: BCVA improvement of five letters or more from baseline.

Techniques: Imaging

Journal: BMC Ophthalmology

Article Title: Predictive effect of TCED-HFV grading and imaging biomarkers on anti-VEGF therapy in diabetic macular edema

doi: 10.1186/s12886-023-02973-7

Figure Lengend Snippet: Mean BCVA EDTR letters in different DME stages before and after 6 months of treatment of anti-VEGF

Article Snippet: Although severe DME staging was an important factor in univariate analysis, it was not included in multivariate analysis because it was composed of other factors included in the model. Fig. 2 Multivariate analysis of imaging biomarkers and outcomes after anti-VEGF therapy in patients with diabetic macular edema (DME). (A) Endpoint 1: CST reduction of 10% or more from baseline. (B) Endpoint 2: CST < 300 μm at 6 months. (C) Endpoint 3: BCVA improvement of five letters or more from baseline.

Techniques:

Journal: BMC Ophthalmology

Article Title: Predictive effect of TCED-HFV grading and imaging biomarkers on anti-VEGF therapy in diabetic macular edema

doi: 10.1186/s12886-023-02973-7

Figure Lengend Snippet: Mean CST in different DME stages before and after 6 months of treatment of anti-VEGF

Article Snippet: Although severe DME staging was an important factor in univariate analysis, it was not included in multivariate analysis because it was composed of other factors included in the model. Fig. 2 Multivariate analysis of imaging biomarkers and outcomes after anti-VEGF therapy in patients with diabetic macular edema (DME). (A) Endpoint 1: CST reduction of 10% or more from baseline. (B) Endpoint 2: CST < 300 μm at 6 months. (C) Endpoint 3: BCVA improvement of five letters or more from baseline.

Techniques:

Journal: Clinical, Cosmetic and Investigational Dermatology

Article Title: EDIL3 and VEGF Synergistically Affect Angiogenesis in Endothelial Cells

doi: 10.2147/CCID.S411253

Figure Lengend Snippet: Results of immunohistochemical assay. EDIL3 and VEGF expression levels were upregulated in psoriatic skin lesions and were correlated with the PASI. ( a ) EDIL3 and VEGF expression levels in psoriatic skin lesions (PP) and normal skin (NN) were determined by immunohistochemical assay using antibodies against EDIL3 and VEGF. The brown section of the photomicrographs indicates the positive expression of EDIL3 or VEGF (magnification: 20×). ( b ) EDIL3 overexpression in psoriatic skin lesions was positively correlated with the PASI ( p < 0.05, r = 0.811). ( c ) VEGF overexpression in psoriatic skin lesions was positively correlated with PASI ( p < 0.05, r = 0.0784).

Article Snippet: The blots were then incubated with rabbit anti-human antibodies against β-actin (CST, USA), EDIL3, VEGF, and VEGFR2 (all from Abcam, UK).

Techniques: Immunohistochemical staining, Expressing, Over Expression

Journal: Clinical, Cosmetic and Investigational Dermatology

Article Title: EDIL3 and VEGF Synergistically Affect Angiogenesis in Endothelial Cells

doi: 10.2147/CCID.S411253

Figure Lengend Snippet: Images of Western blotting assay. ( a ) After EDIL3 knockdown in ECs, the expression of EDIL3 was detected. The image shows that EDIL3 expression in the si-EDIL3 group was significantly lower than that in the control group. ( b ) VEGF and VEGFR2 protein levels in the cells of both groups were determined by Western blotting assay. EDIL3 promoted VEGF and VEGFR2 expression in ECs.

Article Snippet: The blots were then incubated with rabbit anti-human antibodies against β-actin (CST, USA), EDIL3, VEGF, and VEGFR2 (all from Abcam, UK).

Techniques: Western Blot, Expressing

Journal: Heliyon

Article Title: Emodin prevents renal ischemia-reperfusion injury via suppression of p53-mediated cell apoptosis based on network pharmacology

doi: 10.1016/j.heliyon.2023.e15682

Figure Lengend Snippet: Emodin regulated the levels of HIF-1α and VEGF. (A – D) The effect of emodin on the protein levels of HIF-1α and VEGF in renal I/R model rats. I/R: ischemia/reperfusion; β-actin were used as internal control. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Primary antibodies against VEGF and HIF-1α (Cell Signaling Technology, USA) were prepared in advance.

Techniques:

Journal: Research

Article Title: Characteristic Features of Deep Brain Lymphatic Vessels and Their Regulation by Chronic Stress

doi: 10.34133/research.0120

Figure Lengend Snippet: Chronic stress down-regulates VEGF-C signaling pathway. (A) Western blot images of IL-1β and TNF-α in Control and Stress groups. The codetection of β-actin bands served as loading control. (B to D) Quantitative analysis of the 17-kD IL-1β (B), 31-kD IL-1β (C), and TNF-α (D). All data were normalized to the corresponding band of the loading control β-actin. (E) Western blot images of VEGF-C, VEGFR2, and VEGFR3 as well as the cleaved and soluble VEGFR2 (sVEGFR2) in Control and Stress groups. The codetection of β-actin bands served as loading control. (F to I) Quantitative analysis of VEGF-C (F), VEGFR3 (G), VEGFR2 (H), and sVEGFR2 (I, presented as sVEGFR2/VEGFR2 ratio). All data were normalized to the corresponding band of the loading control β-actin. (J) Coimmunoprecipitation of VEGF-C and sVEGFR2 in hippocampal lysates by using VEGF-C antibody. (K) Coimmunoprecipitation of VEGF-C and VEGFR2 or sVEGFR2 in hippocampal lysates obtained from Control or Stress mice by using VEGFR2 and sVEGFR2 antibodies. (L) Quantitative analysis of the fractions of VEGF-C that binds to sVEGR2 as a ratio to that binding to VEGFR2 in Control and Stress groups. Data are presented as means ± SEM. * P < 0.05, ** P < 0.01. For detailed statistical analysis, see Tables to .

Article Snippet: For quantifying the amount of VEGF-C that binds to VEGFR2 and to compare it with that that binds to sVEGFR2, 2 μl Rabbit Monoclonal VEGFR2 Antibody (1:100, C-terminus, Cell Signaling Technology, #2479) or 2 μl of Rabbit Polyclonal VEGFR2 antibody (1:100.

Techniques: Western Blot, Binding Assay

Journal: Research

Article Title: Characteristic Features of Deep Brain Lymphatic Vessels and Their Regulation by Chronic Stress

doi: 10.34133/research.0120

Figure Lengend Snippet: Antibodies source, dilutions, and incubation conditions.

Article Snippet: For quantifying the amount of VEGF-C that binds to VEGFR2 and to compare it with that that binds to sVEGFR2, 2 μl Rabbit Monoclonal VEGFR2 Antibody (1:100, C-terminus, Cell Signaling Technology, #2479) or 2 μl of Rabbit Polyclonal VEGFR2 antibody (1:100.

Techniques: Incubation, Flow Cytometry

Journal: PLOS ONE

Article Title: VEGF-A165 is the predominant VEGF-A isoform in platelets, while VEGF-A121 is abundant in serum and plasma from healthy individuals

doi: 10.1371/journal.pone.0284131

Figure Lengend Snippet: Mixture of rhVEGF-A121, rhVEGF-A165 and rhVEGF-A189 (50 μg each) were subjected to 15% SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE). The transferred membrane was detected with anti-VEGF-A polyclonal antibody (left panel), anti-VEGF-A165 monoclonal antibody (middle panel), and anti-VEGF-A121 monoclonal antibody (right panel). The upper band of VEGF-A121 bands in SDS-PAGE (Lane 3) is due to glycosylation according to manufacturer’s instruction provided from Cell Signaling Technologies.

Article Snippet: Recombinant human VEGF-A121 (rhVEGF-A121) and VEGF-A165 (rhVEGF-A165) were purchased from Cell signaling Technologies (Danvers, MA).

Techniques: Polyacrylamide Gel Electrophoresis, SDS Page

Journal: PLOS ONE

Article Title: VEGF-A165 is the predominant VEGF-A isoform in platelets, while VEGF-A121 is abundant in serum and plasma from healthy individuals

doi: 10.1371/journal.pone.0284131

Figure Lengend Snippet: Specific immunoreactivity of the anti-VEGF-A121 and anti-VEGF-A165 monoclonal antibodies using for ELISA. The absorbance values at 450 nm (Abs 450 nm) obtained by VEGF-A121 ELISA (A, C) and VEGF-A165 ELISA (B, D). For these ELISAs, different doses of rhVEGF-A121, rhVEGF-A165, and rhVEGF-A189 (A, B) or rhVEGF-A206 (C, D) were evaluated. VEGF-A121 ELISA and VEGF-A165 ELISA specifically recognized rhVEGF-A121 (A, C) and rhVEGF-A165 (B, D), respectively.

Article Snippet: Recombinant human VEGF-A121 (rhVEGF-A121) and VEGF-A165 (rhVEGF-A165) were purchased from Cell signaling Technologies (Danvers, MA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLOS ONE

Article Title: VEGF-A165 is the predominant VEGF-A isoform in platelets, while VEGF-A121 is abundant in serum and plasma from healthy individuals

doi: 10.1371/journal.pone.0284131

Figure Lengend Snippet: (A, B) A standard curve of rhVEGF-A121 by VEGF-A121 ELISA (A) and a standard curve of rhVEGF-A165 by VEGF-A165 ELISA (B) were independently created. (C, D) Different lower detection limits for rhVEGF-A121 and rhVEGF-A165 were determined in each ELISA (n = 5); the ELISA lower detection limits for VEGF-A121 or VEGF-A165 were 35 pg/mL (C) or 38 pg/mL (D), respectively. Abs 450 nm represents absorbance at 450 nm.

Article Snippet: Recombinant human VEGF-A121 (rhVEGF-A121) and VEGF-A165 (rhVEGF-A165) were purchased from Cell signaling Technologies (Danvers, MA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLOS ONE

Article Title: VEGF-A165 is the predominant VEGF-A isoform in platelets, while VEGF-A121 is abundant in serum and plasma from healthy individuals

doi: 10.1371/journal.pone.0284131

Figure Lengend Snippet: (A) HEK293 cells were transfected with plasmids overexpressed VEGF-A121 (pVEGF-A121) and VEGF-A165 (pVEGF-A165) and empty plasmid (mock). Their cell lysates were blotted by VEGF-A polyclonal antibody and β-actin. (B—D) Total VEGF-A in the media from (A) was measured by Human VEGF Quantikine ELISA Kit (B), VEGF-A121 ELISA (C), and VEGF-A165 ELISA (D). Graph represents mean ± SD; n = 9.

Article Snippet: Recombinant human VEGF-A121 (rhVEGF-A121) and VEGF-A165 (rhVEGF-A165) were purchased from Cell signaling Technologies (Danvers, MA).

Techniques: Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: PLOS ONE

Article Title: VEGF-A165 is the predominant VEGF-A isoform in platelets, while VEGF-A121 is abundant in serum and plasma from healthy individuals

doi: 10.1371/journal.pone.0284131

Figure Lengend Snippet: Concentrations of total VEGF-A, VEGF-A121, and VEGF-A165 were measured in serum and plasma from healthy volunteers (n = 20). Correlation between total VEGF-A and VEGF-A121 or VEGF-A165 in serum (A) and plasma (B) were evaluated by a linear regression analysis (left panels of A and B: total VEGF-A vs. VEGF-A121, right panels of A and B: total VEGF-A vs. VEGF-A165). Correlations were determined using Spearman’s correlation test (two-tailed). Results are represented as significant at p < 0.05.

Article Snippet: Recombinant human VEGF-A121 (rhVEGF-A121) and VEGF-A165 (rhVEGF-A165) were purchased from Cell signaling Technologies (Danvers, MA).

Techniques: Two Tailed Test

Journal: PLOS ONE

Article Title: VEGF-A165 is the predominant VEGF-A isoform in platelets, while VEGF-A121 is abundant in serum and plasma from healthy individuals

doi: 10.1371/journal.pone.0284131

Figure Lengend Snippet: (A, B) VEGF-A121 and VEGF-A165 in plasma and serum (A) and those in platelet lysate (PL-VEGF-A121 and PL-VEGF-A165) (B) were measured by isoform specific ELISAs (n = 59). (C) VEGF-A121 released from aggregating platelets (PR-VEGF-A121) and VEGF-A121 in platelet lysate (PL-VEGF-A121) were the same levels for 10 6 number of platelets (left panel). VEGF-A165 released from aggregating platelets (PR-VEGF-A165) was significantly lower than VEGF-A165 in platelet lysate (PL-VEGF-A165) (right panel) (n = 8). (D) The ratio of PR-VEGF-A121 to PL-VEGF-A121 and that of PR-VEGF-A165 to PL-VEGF-A165 in the same number of platelets were calculated from (C). *P < 0.0001, **P < 0.05, and ***P < 0.001.

Article Snippet: Recombinant human VEGF-A121 (rhVEGF-A121) and VEGF-A165 (rhVEGF-A165) were purchased from Cell signaling Technologies (Danvers, MA).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: L-carnitine prevents lenvatinib-induced muscle toxicity without impairment of the anti-angiogenic efficacy

doi: 10.3389/fphar.2023.1182788

Figure Lengend Snippet: Antibodies used for Western blotting.

Article Snippet: VEGF Receptor 2 (55B11) , Cell Signaling 2479 , 1:1,000.

Techniques: Western Blot

Journal: Frontiers in Pharmacology

Article Title: L-carnitine prevents lenvatinib-induced muscle toxicity without impairment of the anti-angiogenic efficacy

doi: 10.3389/fphar.2023.1182788

Figure Lengend Snippet: Antibodies used for Western blotting.

Article Snippet: Phospho-VEGF Receptor 2 (Tyr1175) , Cell Signaling 2478 , 1:1,000.

Techniques: Western Blot

Journal: Clinical Epigenetics

Article Title: Neuroglobin plays as tumor suppressor by disrupting the stability of GPR35 in colorectal cancer

doi: 10.1186/s13148-023-01472-2

Figure Lengend Snippet: NGB-OE suppresses tumor angiogenesis ability of CRC cells. A Heatmap of differentially expressed proteins in NGB-overexpressing or vector HCT116 cells. B Angiogenesis estimated by endothelial cell tube formation assay; statistical analysis of tube formation and branches is shown on the right used angiogenesis tool of image J, N = 3. CM means conditional medium. C Immunofluorescence: the effect of NGB on CD31 expression status in CRC cells. D The status of angiogenesis pathway detected by WB or WES. E Immunofluorescence: the effect of NGB on VEGFR2 expression in CRC cells. All experiments were performed in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Antibodies against p-VEGFR2 (#2478), pAKT (#4060), p-Src (#6943) and p-ERK 1/2 (#4370) were purchased from CST.

Techniques: Plasmid Preparation, Endothelial Cell Tube Formation Assay, Immunofluorescence, Expressing

Journal: bioRxiv

Article Title: Regulation of VEGFR2 and AKT signaling by Musashi-2 in lung cancer

doi: 10.1101/2023.03.29.534783

Figure Lengend Snippet: MSI2 regulation of VEGFR2 protein level in a mouse cell line. (A) Heatmap of RPPA results for VEGFR2 protein expression in 344SQ cell line transfected with empty vector or shRNAs or MSI2 depletion with two independent shRNAs (M2-m1 and M2-m2). (B) Western blots of 344SQ cell line lysates, following depletion (MSI2 KD sh1, sh2) and overexpression (MSI2 OE, MSI2) of MSI2. pLKO and pLV are negative controls. MSI2 depletion was induced by the addition of 1 μg/ml of Doxycycline for 48 h. (C) Quantification of Western blot data from at least three independent experiments by Image J software, with values normalized to negative control and β-actin. (D) RT-qPCR analysis of Vegfr2 and Vegf-a mRNA from 344SQ cell line following depletion (MSI2 KD, sh1, sh2) and overexpression (MSI2 OE, MSI2) of MSI2. pLKO and pLV are negative controls. MSI2 depletion was induced by the addition of 1 μg/ml of Doxycycline for 48 h. Data normalized to negative control and Polr2a. Error bars represented by SEM. Statistical analysis was performed using an unpaired two-tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001 for all graphs.

Article Snippet: Anti-MSI2 (#ab76148), anti-VEGF-A (#ab46154) were obtained from Abcam (Cambridge, UK), anti-VEGFR2 (#2479), anti-phVEGFR2 (#3817) anti-β-actin (#3700), anti-phAKT (T308) (#13038), anti-phAKT (S473) (#4060), anti-AKT total (#2920), anti-phGSKαβ (#9331), anti-GSKαβ total (#5676), anti-PTEN (#14642), anti-phERK (#4370), anti-ERK total (4695), anti-ph4EBP(#2855), anti-phP70SK (#9206), anti-P70S6K total (#2708) anti-rabbit HRP-linked (#7074), anti-mouse HRP-linked (#7076) were obtained from Cell Signaling (Danvers, MA).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Over Expression, Software, Negative Control, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: Regulation of VEGFR2 and AKT signaling by Musashi-2 in lung cancer

doi: 10.1101/2023.03.29.534783

Figure Lengend Snippet: (A) Western blot of human NSCLC cell lines. (B) RT-qPCR analysis of VEGFR2 mRNA from human NSCLC cell lines. Data from at least three independent experiments by Image J software and normalized to 18S rRNA and to A549. (C) MSI2 consensus binding sites in human mRNAs. Location of consensus binding sites for Musashi proteins in the noted human genes, as defined from studies by Bennett et al 1 , and Wang et al 2 . Coding sequences are represented by thick lines; 3’ untranslated regions by a thin line. 7- or 8-bp consensus sequences are indicated by arrows. VEGFR2 reference sequence - NCBI Reference Sequence: NM_002253.4, PTEN reference sequence - NCBI Reference Sequence: NM_000314.8. (D) Quantification of mRNA immunoprecipitation (RIP) results from assays performed in Hcc1171 and H441 cell lysates using antibodies to MSI2, or IgG (negative control) antibodies, followed by quantitative RT-PCR. Data are normalized to positive control PTP4A1, TGFBR1 , and SMAD3 are additional positive controls; GAPDH and ACTB are negative control. The data shown reflect the average of three independent RIP experiments. Error bars represented by SEM. Statistical analysis was performed using an unpaired two-tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001 for all graphs.

Article Snippet: Anti-MSI2 (#ab76148), anti-VEGF-A (#ab46154) were obtained from Abcam (Cambridge, UK), anti-VEGFR2 (#2479), anti-phVEGFR2 (#3817) anti-β-actin (#3700), anti-phAKT (T308) (#13038), anti-phAKT (S473) (#4060), anti-AKT total (#2920), anti-phGSKαβ (#9331), anti-GSKαβ total (#5676), anti-PTEN (#14642), anti-phERK (#4370), anti-ERK total (4695), anti-ph4EBP(#2855), anti-phP70SK (#9206), anti-P70S6K total (#2708) anti-rabbit HRP-linked (#7074), anti-mouse HRP-linked (#7076) were obtained from Cell Signaling (Danvers, MA).

Techniques: Western Blot, Quantitative RT-PCR, Software, Binding Assay, Sequencing, Immunoprecipitation, Negative Control, Positive Control, Two Tailed Test

Journal: bioRxiv

Article Title: Regulation of VEGFR2 and AKT signaling by Musashi-2 in lung cancer

doi: 10.1101/2023.03.29.534783

Figure Lengend Snippet: Consequences of MSI2 depletion (left) and overexpression (right) on mRNA expression levels of VEGFR2 and VEGF-A. Quantitative RT-PCR of mRNA of human NSCLC cell lines, following MSI2 depletion by shRNA (sh1, sh2) and siRNA (si1, si2) and overexpression (MSI2) of MSI2. Negative controls include pLKO, SCR, pLV. Error bars represented by SEM. Statistical analysis was performed using an unpaired two-tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Anti-MSI2 (#ab76148), anti-VEGF-A (#ab46154) were obtained from Abcam (Cambridge, UK), anti-VEGFR2 (#2479), anti-phVEGFR2 (#3817) anti-β-actin (#3700), anti-phAKT (T308) (#13038), anti-phAKT (S473) (#4060), anti-AKT total (#2920), anti-phGSKαβ (#9331), anti-GSKαβ total (#5676), anti-PTEN (#14642), anti-phERK (#4370), anti-ERK total (4695), anti-ph4EBP(#2855), anti-phP70SK (#9206), anti-P70S6K total (#2708) anti-rabbit HRP-linked (#7074), anti-mouse HRP-linked (#7076) were obtained from Cell Signaling (Danvers, MA).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, shRNA, Two Tailed Test

Journal: bioRxiv

Article Title: Regulation of VEGFR2 and AKT signaling by Musashi-2 in lung cancer

doi: 10.1101/2023.03.29.534783

Figure Lengend Snippet: (A) Western blots of indicated cell lines, following depletion by shRNA (sh1, sh2) and siRNA (si1, si2) of MSI2. Negative controls include pLKO and SCR. (B) Quantification of Western blot data from at least three independent experiments by Image J software, with values normalized to negative control and β-actin. (C) Rescue experiment: western blots of A549 and H441 cell lines, following depletion by shRNA (sh1) of MSI2 and VEGFR2 overexpression (VEGFR2 OE). Negative control is pHAGE. (D) Quantification of Western blot data from at least three independent experiments by Image J software, with values normalized to empty vector and β-actin. (E) Western blot of indicated cell lines, following depletion by shRNA (sh1) of MSI2 and VEGFR2 overexpression (VEGFR2 OE). (F) Quantification of Western blot data from at least three independent experiments by Image J software, with values normalized to empty vector and β-actin. Error bars represented by SEM. Statistical analysis was performed using an unpaired two-tailed t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p <0.0001.

Article Snippet: Anti-MSI2 (#ab76148), anti-VEGF-A (#ab46154) were obtained from Abcam (Cambridge, UK), anti-VEGFR2 (#2479), anti-phVEGFR2 (#3817) anti-β-actin (#3700), anti-phAKT (T308) (#13038), anti-phAKT (S473) (#4060), anti-AKT total (#2920), anti-phGSKαβ (#9331), anti-GSKαβ total (#5676), anti-PTEN (#14642), anti-phERK (#4370), anti-ERK total (4695), anti-ph4EBP(#2855), anti-phP70SK (#9206), anti-P70S6K total (#2708) anti-rabbit HRP-linked (#7074), anti-mouse HRP-linked (#7076) were obtained from Cell Signaling (Danvers, MA).

Techniques: Western Blot, shRNA, Software, Negative Control, Over Expression, Plasmid Preparation, Two Tailed Test

Journal: bioRxiv

Article Title: Regulation of VEGFR2 and AKT signaling by Musashi-2 in lung cancer

doi: 10.1101/2023.03.29.534783

Figure Lengend Snippet: (A) Representative IHC images of MSI2 and VEGFR2 expression in normal lung and lung tumors. (B) MSI2 and VEGF-A (n=94), MSI2 and VEGFR2 (n=118) H-score correlation of human NSCLC TMAs. For IHC quantification, each spot was examined by board-certified pathologists (ED and NK) who assigned a score of 0 (no staining), 1+ (weak staining), 2+ (moderate staining), and 3+ (strong staining) within carcinomatous areas. The score for each of the two tumor spots was averaged for statistical analysis. The H-score, which ranges from 0 to 300, was calculated using the following formula: [1x(% cells 1+) + 2x(% cells 2+) + 3x(% cells 3+)], which reflects staining intensity as well as percentage of positive cells. A sum of p2 and p3 represents a sum of 2+ and 3+ cells (2x (% cells 2+) + 3x(% cells 3+), which excludes 1+ cells.

Article Snippet: Anti-MSI2 (#ab76148), anti-VEGF-A (#ab46154) were obtained from Abcam (Cambridge, UK), anti-VEGFR2 (#2479), anti-phVEGFR2 (#3817) anti-β-actin (#3700), anti-phAKT (T308) (#13038), anti-phAKT (S473) (#4060), anti-AKT total (#2920), anti-phGSKαβ (#9331), anti-GSKαβ total (#5676), anti-PTEN (#14642), anti-phERK (#4370), anti-ERK total (4695), anti-ph4EBP(#2855), anti-phP70SK (#9206), anti-P70S6K total (#2708) anti-rabbit HRP-linked (#7074), anti-mouse HRP-linked (#7076) were obtained from Cell Signaling (Danvers, MA).

Techniques: Expressing, Staining