Journal: Nature cardiovascular research

Article Title: Sinusoidal and lymphatic vessel growth is controlled by reciprocal VEGF-C–CDH5 inhibition

doi: 10.1038/s44161-022-00147-0

Figure Lengend Snippet: a, Immunoblot analysis and quantification of pY685 VE-cadherin and pY418 SRC in response to treatment VEGF-C or the VEGFR3-specific VEGF-CC156S for 15 minutes. n=3 independent experiments per condition. b, Immunofluorescence staining and quantification for VE-cadherin (red) and pY418 Src (green) in human LECs after treatment with VEGF-C for 15 minutes. Bottom panels show colocalized VE-cadherin and pY418 SRC. n=3–4 independent experiments per condition. c, Immunofluorescence staining for pY418 SRC (green) and Endomucin (red) in bone marrow sections from Control and Cdh5CreERT2; Flt4fl/fl mice, and quantification of pY418 SRC FI normalized to Endomucin+ area. Control, n=5. Cdh5CreERT2; Flt4fl/fl, n=4. d, Immunoblot analysis and quantification of pY418 SRC in human LECs after knockdown of VEGFR3 and VE-cadherin and treatment with VEGF-C for 15 minutes. n=3 independent experiments per condition. e, Immunoblot analysis and quantification of pERK1/2 and pY685 VE-cadherin expression in response to VEGF-C with the SRC inhibitor SU6656 or MEK1/2 inhibitor U0126. n=3 independent experiments per condition. f, Immunofluorescence staining and quantification of human LECs that were non-treated (NT) or treated for VEGF-C for 30 minutes with or without SU6656 for the early endosome marker EEA1 (green) and VE-cadherin (red). Yellow arrows denote EEA1+VE-cadherin+ endosomes. n=16–19 fields of view from 3 independent experiments per condition. Scale bars = 25μm. g, h, Immunofluorescence staining of bone marrow from E18.5 Control and R26CreERT2; Vegfcfl/fl (g, Control, n=7. R26CreERT2; Vegfcfl/fl, n=7) or Cdh5CreERT2; Flt4fl/fl (h, Control, n=5. Cdh5CreERT2; Flt4fl/fl, n=4) littermates for Endomucin (red) and VE-cadherin (green). Boxes in the left image denote area of magnified image in gray scale below. Quantification of VE-cadherin fold change measured by VE-cadherin fluorescence intensity (FI) normalized to Endomucin+ area. Scale bars = 100μm. i, Immunofluorescence staining of human LECs that were treated with VEGF-C for 30 minutes with knockdown of VE-cadherin for EEA1 (red) and VEGFR3 (green). Yellow arrows denote EEA1+VEGFR3+ endosomes. n=50 fields of view from 4 independent experiments per condition. Scale bars = 10μm. j, Immunofluorescence staining of human LECs that were treated with VEGF-C for 30 minutes with knockdown of VEGFR3 and VE-cadherin for EEA1 (red) and VEGFR2 (green). Yellow arrows denote EEA1+VEGFR2+ endosomes. n=21 fields of view from 3 independent experiments per condition. Scale bars = 10μm. Statistical analysis was performed using two-tailed, unpaired Welch’s t-test for a, b, c, e, g, h, and i and one-way ANOVA with Tukey’s test for multiple comparisons for d, f, and j. Data are shown as means±S.D. or median±1st/3rd quartile for violin plots.

Article Snippet: Antibodies for immunoblot: pERK1/2 (1:2000, Cell Signaling Technology 4370), total ERK (1:1000, Cell Signaling Technology 4695), pY685 VE-cadherin (1:500, Millipore Sigma ABT1760), human VE-cadherin (1:1000 R&D AF938), pAKT (1:1000, Cell Signaling Technology 4060), total AKT (1:1000, Cell Signaling Technology 4685), pS6 (1:1000, Cell Signaling Technology 4858), total S6 (1:1000, Cell Signaling Technology 2317), pY418 SRC (1:500, Abcam ab133460), total SRC (1:1000, Cell Signaling Technology 2109), human VEGFR3 (1:1000, Millipore Sigma MAB3757), β-actin (1:2000, Abcam ab8226).

Techniques: Western Blot, Immunofluorescence, Staining, Expressing, Marker, Fluorescence, Two Tailed Test

Journal: Journal of Signal Transduction

Article Title: Podocyte Protein, Nephrin, Is a Substrate of Protein Tyrosine Phosphatase 1B

doi: 10.1155/2011/376543

Figure Lengend Snippet: PTP-PEST decreases nephrin phosphorylation via Src-family kinase. (a) Pull-down was performed as in with additional plasmids of GST-PTP-PEST-WT and GST-PTP-PEST-CS (substrate-trapping mutant). The substrate-trapping mutant of PTP1B, but not of PTP-PEST, pulled down nephrin. (b) Cos-1 cells were transiently cotransfected with pEBG (empty vector), GST-PTP-PEST-WT, or GST-PTP-PEST-CS (substrate trapping mutant) and fyn. After 24 hrs, cell lysates were subjected to GST pull down as in or to immunoprecipitation for fyn. Only the substrate-trapping mutant of PTP-PEST (CS), but not the wild-type, pulled down fyn or was coimmunoprecipitated with fyn. fyn, which was pulled down by PTP-PEST-CS, was strongly phosphorylated at pY418, suggesting that pY418 containing region is the substrate of PTP-PEST. Wild-type PTP-PEST decreased pY418-Src in total lysates. Note that pY418 antibody cross-reacts to all the Src-family kinases.

Article Snippet: Antibodies were purchased from the following sources: PTP1B and SHP2 (BD Biosciences), PTP-PEST (Cell Signaling), α -tubulin (Abcam), 4G10 (for pY, Millipore), GST and fyn (Santa Cruz Biotech.), pY418-Src (Invitrogen).

Techniques: Mutagenesis, Plasmid Preparation, Immunoprecipitation