Journal: PLoS ONE

Article Title: HIF-1 and c-Src Mediate Increased Glucose Uptake Induced by Endothelin-1 and Connexin43 in Astrocytes

doi: 10.1371/journal.pone.0032448

Figure Lengend Snippet: A ) Astrocytes were incubated in the absence (control, C) or presence of 0.1 µM ET-1 for the indicated times, proteins were extracted and Y416 c-Src and total c-Src were analysed by Western blot. The results are expressed as percentages of the level found in the control and they show that ET-1 rapidly and transiently increased Y416 c-Src without affecting significantly total c-Src. ***p<0.001; *p<0.05 versus control. B ) Astrocytes were transfected with NT-siRNA or with Cx43-siRNA. After 48 h, proteins were extracted and Y416 c-Src, total c-Src and Cx43 were analysed by Western blot. The results are expressed as percentages of the level found in the control and they show that silencing Cx43 increased Y416 c-Src without affecting significantly total c-Src. **p<0.01 versus NT-siRNA.

Article Snippet: Rabbit polyclonal antibody against total c-Src (2108) and rabbit polyclonal antibody against Y416 c-Src (2101) were from Cell Signaling Technology (Boston, EEUU).

Techniques: Incubation, Western Blot, Transfection

Journal: PLoS ONE

Article Title: HIF-1 and c-Src Mediate Increased Glucose Uptake Induced by Endothelin-1 and Connexin43 in Astrocytes

doi: 10.1371/journal.pone.0032448

Figure Lengend Snippet: Astrocytes were preincubated with 100 ng/µL PP2 (c-Src inhibitor) or 100 ng/µL PP3 (inactive analogue) for 1 h and these agents were maintained for the rest of the experiment. Then, cells were incubated in the absence or presence of 0.1 µM ET-1 for the indicated times and proteins were extracted for the analysis of Y416 c-Src and total c-Src by Western blot. The results are expressed as percentages of the level found in the control (PP3, time 0) and they show that PP2 inhibited the activation of c-Src (Y416 c-Src) promoted by ET-1. ***p<0.001; *p<0.05 versus the corresponding time 0.

Article Snippet: Rabbit polyclonal antibody against total c-Src (2108) and rabbit polyclonal antibody against Y416 c-Src (2101) were from Cell Signaling Technology (Boston, EEUU).

Techniques: Incubation, Western Blot, Activation Assay

Journal: PLoS ONE

Article Title: c-Src kinase inhibits osteogenic differentiation via enhancing STAT1 stability

doi: 10.1371/journal.pone.0241646

Figure Lengend Snippet: (A) c-Src activity during 21 days of osteogenic differentiation in mESCs. p-Y416-c-Src level and total level of c-Src are analyzed by immunoblotting. GAPDH served as loading control. (B) p-Y416-c-Src band densities are quantified using ImageJ. Normalized values to corresponding total c-Src and GAPDH controls are graphed. (C) Total RNA of day 21 osteo-nodules is subjected to qPCR analysis using OC primer pairs. Fold change in OC expression is calculated by normalizing PP2 treated values to their corresponding PP3 control. Values representing the mean (±SD) of triplicates are graphed and subjected to one-way ANOVA; F (15,32) = 15.85, p <0.0001. Bonferroni's multiple comparisons test indicated significant differences between PP2 and PP3 treated cells for day 6–10 ( p <0.0001), day 10–17 ( p = 0.0161), and day 10–21 ( p = 0.0422). (D) Day 21 osteo-nodules were stained by ARS method. Absorbed stains were extracted and their concentration were quantified and normalized to their corresponding PP3 controls. Data shown represent the mean (±SD) of triplicates and subjected to one-way ANOVA; F (15,32) = 8.651, p <0.0001. Bonferroni's multiple comparisons test revealed significant differences between PP2 and PP3 treated cells for day 6–10 with p <0.0001. (E) Day 14 qPCR analysis for OC mRNA expression in MC3T3-E1s upon overexpression of mt c-Src. Values from doxycycline- free condition were used to normalize the induced condition. Data representing mean of triplicate values (±SD) are graphed. (F) Day 14 differentiated MC3T3-E1 cells for the indicated conditions were subjected to ARS for mineralization assessment and concentrations of extracted Alizarin Red S were quantified. Mean of triplicates (±SD) are graphed and subjected to one-way ANOVA; F (3,8) = 3066, p <0.0001. Bonferroni post hoc analysis was **** p <0.0001 for the indicated comparisons.

Article Snippet: For immunoblotting, anti-STAT1 (Cell Signaling, catalog No.9172S), anti-GAPDH (Cell Signaling, Catalog No.2118S), anti-c-Src (Cell Signaling, Catalog No. 2108), anti-p-Y416-c-Src (Cell Signaling, Catalog No.2101S), anti- Runx2 (Cell Signaling, Catalog No.8486S) antibodies were applied.

Techniques: Activity Assay, Western Blot, Expressing, Staining, Concentration Assay, Over Expression

Journal: PLoS ONE

Article Title: c-Src kinase inhibits osteogenic differentiation via enhancing STAT1 stability

doi: 10.1371/journal.pone.0241646

Figure Lengend Snippet: (A and B) Real time qPCR analysis of STAT1 expression upon pharmacological inhibition of c-Src using 10 μM of PP1, SrcI-1, and PP2 for 2 hours where DMSO served as negative control (A) and c-Src downregulation using with three different distinct siRNAs where silencer™ select negative siRNA served as control for 48 hours (B) in MC3T3E1 cells. Data shown represent the means (± SD) of triplicates. (C) c-Src inhibition and its effect on STAT1 protein level in mESCs. Differentiating EBs were exposed to 10 μM of PP2 or PP3 for 24 hours prior to treatment with CHX (10 μM) or DMSO. Lysates were collected at the indicated times and subjected to WB analysis using STAT1 antibody. GAPDH served as loading control. (D) Inhibition of c-Src activity and its effect on STAT1 half-life in MC3T3-E1 cells. Cells were subjected to PP2 (10 μM) or DMSO (solvent ctrl) treatments for 2 hrs, prior to addition of CHX (10 uM). Harvested cells at the indicated time points were subjected to WB analysis to examine STAT1 expression. GAPDH served as a loading control. (E) Quantification of STAT1 stability assays. STAT1 band densities were quantified, first normalized to the corresponding GAPDH and then to t = 0 DMSO controls. (F and G) Inhibition of proteasomal degradation by MG132 and its effect on STAT1 loss resulted from c-Src inhibition. MC3T3-E1s were treated with three different c-Src inhibitors including PP2, Src I-1, and PP1 (10 μM) for 24 hrs (F) or were transfected with control siRNA or different combinations of three different specific c-Src siRNA for 48 hrs (A = c-Src siRNAs #1+ #2, B = c-Src siRNAs #1+ #3, and C = c-Src siRNAs #2+ #3) (G). Before harvest, cells were treated with MG132 (10 μM) for 4 hrs as indicated. STAT1, c-Src, and p-c-Src Y416 protein levels were analyzed by immunoblots, with GAPDH as a loading control. (H) Inhibition of c-Src activity and its effect on STAT1 ubiquitination. Proteasomal degradation was inhibited by MG132 in MC3T3-E1 cells for two hours. DMSO served as the negative control. After two hours treated cells underwent second treatments with PP2 (10 μM) or DMSO for another 2 hours. Lysates were subjected to IP assay using STAT1 antibody. Precipitated immunocomplexes were then analyzed by WB using both STAT1 antibody.

Article Snippet: For immunoblotting, anti-STAT1 (Cell Signaling, catalog No.9172S), anti-GAPDH (Cell Signaling, Catalog No.2118S), anti-c-Src (Cell Signaling, Catalog No. 2108), anti-p-Y416-c-Src (Cell Signaling, Catalog No.2101S), anti- Runx2 (Cell Signaling, Catalog No.8486S) antibodies were applied.

Techniques: Expressing, Inhibition, Negative Control, Activity Assay, Transfection, Western Blot

Journal: Cell reports

Article Title: A Tyrosine Switch on NEDD4-2 E3 Ligase Transmits GPCR Inflammatory Signaling

doi: 10.1016/j.celrep.2018.08.061

Figure Lengend Snippet:

Article Snippet: Rabbit anti-phospho-c-Src (Y416) , Cell signaling Technology , Cat# 2101; RRID:AB_331697.

Techniques: Recombinant, Sequencing, Negative Control, Software