Journal: Journal of Clinical Investigation

Article Title: uPAR isoform 2 forms a dimer and induces severe kidney disease in mice

doi: 10.1172/jci124793

Figure Lengend Snippet: Figure 7. Blocking Src activity reduces proteinuria in msuPAR2-Tg mice. (A) Glomerular Src activity was determined by immunofluorescent staining of the kidney cryosections with p-Src monoclonal antibody (green). Podocin was used as a podocyte marker (red). Shown are the representative glomeruli with p-Src immunofluorescent staining. As indicated by the p-Src staining intensity, glomerular Src phosphorylation was readily observed in the kidney sections from msuPAR2-Tg, but not from other investigated mice. Scale bars: 20 μm. (B) Treatment with Src inhibitors lowered proteinuria in msuPAR2-Tg mice. Both PP1 and Src inhibitor 1 (5 mg/kg) were administered into the randomly grouped msuPAR2-Tg mice intraperitoneally, with vehicle controls receiving the same amount of DMSO. n = 8 Src inhibitor group; n = 7 vehicle control group. At 72 hours after treatment, proteinuria was significantly reduced by Src inhibitor treatment. Two-way ANOVA; data were log-transformed to normal distribution. **P < 0.01.

Article Snippet: Src phosphorylation was labeled with p–c-Src antibody (A96, 1:100, Santa Cruz Biotechnology Inc.).

Techniques: Blocking Assay, Activity Assay, Staining, Marker, Phospho-proteomics, Control, Transformation Assay

Journal: Journal of Clinical Investigation

Article Title: uPAR isoform 2 forms a dimer and induces severe kidney disease in mice

doi: 10.1172/jci124793

Figure Lengend Snippet: Figure 8. Glomerular c-Src activity is increased in human FSGS kidney. Immunofluorescent staining with p–c-Src antibody was performed for the frozen sections of deidentified human kidney biopsies. Synaptopodin was used as a podocyte marker. Shown is a representative of 4 batches of immu- nostaining. While a minimal amount of c-Src phosphorylation was observed in the glomeruli of healthy donors (n = 3), glomerular p–c-Src intensity was increased in 6 out of 10 FSGS patients. Overlap of p–c-Src (green) and synpo (red ) indicates that p–c-Src was local- ized in podocytes. Of note, only 11 out of 24 observed glomeruli were positive for p–c-Src, from which 64% were focal, 36% globally but not evenly. In contrast, the increase of pSrc was not observed in other glomerular diseases, including SLE (n = 2), MPGN (n = 2), and MCD (n = 4). Scale bar: 20 μm. NT, normal kidney tissue.

Article Snippet: Src phosphorylation was labeled with p–c-Src antibody (A96, 1:100, Santa Cruz Biotechnology Inc.).

Techniques: Activity Assay, Staining, Marker, Phospho-proteomics

Journal: Journal of Cell Science

Article Title: A unique role for clathrin light chain A in cell spreading and migration

doi: 10.1242/jcs.224030

Figure Lengend Snippet: CLCa depletion significantly inhibits spreading-induced signaling. (A) siRNA-transfected cells held in suspension for 1 h (left panel) or plated on collagen IV-coated dishes for the indicated times (minutes, right panel) were lysed and subjected to western blotting with anti-active FAK [pFAK(Y397)], active Src [pSrc(Y416)] and anti-phosphorylated paxillin [pPax(Y118)] antibodies. (B) Protein phosphorylation in control cells at 30 min after plating was set as 100%. The results represent a summary from five to seven experiments. *P<0.05; **P<0.01. (C) Lysates from plated cells treated as in A were analyzed by western blotting with antibodies against Src-dependent FAK phosphorylation sites (Y576 and Y925). The blots shown represent one of three independent experiments.

Article Snippet: Antibodies against the following proteins were used: CLCa (1:1000, sc-28276), CLCb (1:500, sc-376414), actin (1:1000, sc-1616) from Santa Cruz Biotechnology, FAK (1:2000, 610088) and β1-integrin (1:1000, 610467) from BD Transduction Labs, phosphorylated FAK(Y397) (1:1000, 44-624G), phosphorylated paxillin(Y118) (1:1000, 44-722G) from Fisher Scientific, Src (1:2000, 2108), phosphorylated Src(Y416) (1:1000, MAB2685, 2101), phosphorylated FAK(Y576) (1:1000, 3281), FAK(Y925) (1:1000, 3284) from Cell Signaling, phosphorylated Src(Y416) (1:1000, MAB2685) from RD Systems, WAVE1/Scar (1:1000, 07-037), Rac1 (1:2000, 05-389) from Millipore.

Techniques: Transfection, Western Blot

Journal: bioRxiv

Article Title: An allosteric switch between the activation loop and a c-terminal palindromic phospho-motif controls c-Src function

doi: 10.1101/2022.10.16.512342

Figure Lengend Snippet: Kinetics of c-Src auto-phosphorylation. (A) Schematic diagram of the functional domains and auto-phosphorylation sites of c-Src. Surface representation of c-Src in closed autoinhibited and open (active) states, current paradigm for c-Src activation and regulation. (B) WB of samples from a time-course auto-phosphorylation experiment with c-Src WT (3D-construct, 1 μM) in the presence of ATP (1 mM) and MgCl 2 (2 mM) for 0–90 min using the indicated antibodies. Total amount of protein was visualized by Coomassie staining. Phospho-tyrosine quantification (total), data represent mean ± SEM of 6 experiments (n=6). (C) Enzymatic assay performed with c-Src WT (3D-construct, 1 μM) incubated with increasing concentrations of ATP at a fixed concentration (1.5 mg/ml) of c-Src Y419 (IEDNEYTARQG) or Y530 (STEPQYQPGEN) derived peptides. Data represent the mean ± SEM, of 2 experiments (n=2) in duplicate. Enzymatic activity (ODs -1 x 10 −3 ). Catalytic efficiency constants (k cat /K M , fold difference) are depicted in the panel below. (D) Mass spectrum of the [M + 2H] +2 ion (m/z 772.3) of a peptide phosphorylated on Tyr 530 (90 min). Below, phosphorylation kinetics of Tyr419 and Tyr530 phospho-peptides measured by mass spectrometry (0-90 min) are depicted. Data represent the mean of normalized phospho-signal ± SEM of 2 experiments (n=2), with two technical replicates each.

Article Snippet: Antibodies used were: phospho-Src Tyr419 (D49G4, CST #6943), Src (36D10, CST #2109) phospho-Src Tyr 530 (ThermoFisher 44-662G) and total phospho-Tyr (p-Tyr-100 CST #9411) were diluted at 1:10000-1:5000.

Techniques: Functional Assay, Activation Assay, Construct, Staining, Enzymatic Assay, Incubation, Concentration Assay, Derivative Assay, Activity Assay, Mass Spectrometry

Journal: bioRxiv

Article Title: An allosteric switch between the activation loop and a c-terminal palindromic phospho-motif controls c-Src function

doi: 10.1101/2022.10.16.512342

Figure Lengend Snippet: Activation-loop Tyr 419 controls c-Src susbtrate specificity. (A) WB of samples from a time-course auto-phosphorylation experiment with c-Src WT, Y419F and Y530F (3D-construct, 1 μM) in the presence of ATP (1 mM) and MgCl 2 (2 mM) for 0–60 min using the indicated antibodies. Total amount of protein was visualized by Coomassie staining. Phospho-signal quantification (total phospho-tyrosine), data represent mean ± SEM of 6 experiments (n=6) in duplicate. (B) Enzymatic assay performed with c-Src WT, Y419F and Y530F (3D-construct, 1 μM) incubated with increasing concentrations of ATP at a fixed concentration (4 mg/ml) of peptide: ABL derived-peptide (EAIYAAPFAKKK) or RET activation loop Y905 derived-peptide (DVYEEDSFVK). Data represent the mean ± SEM, of 4-6 experiments (n =4-6) in duplicate. Catalytic efficiency constants (k cat /K M , fold difference) are depicted in the right panel. (C-F) WB of samples from a time course phosphorylation assays with c-Src (3D, 1 μM) WT, Y419F and Y530F and using the following substrates surrogates: FAK (aa 1-405), c-Src KD K298M, RET KD (aa 713-1012) K758M and deltaKIF5B-RET, respectively using the indicated antibodies. (G) Logo consensus sequence for optimal c-Src susbtrate, from PhosphoSitePlus (CST).

Article Snippet: Antibodies used were: phospho-Src Tyr419 (D49G4, CST #6943), Src (36D10, CST #2109) phospho-Src Tyr 530 (ThermoFisher 44-662G) and total phospho-Tyr (p-Tyr-100 CST #9411) were diluted at 1:10000-1:5000.

Techniques: Activation Assay, Construct, Staining, Enzymatic Assay, Incubation, Concentration Assay, Derivative Assay, Sequencing

Journal: bioRxiv

Article Title: An allosteric switch between the activation loop and a c-terminal palindromic phospho-motif controls c-Src function

doi: 10.1101/2022.10.16.512342

Figure Lengend Snippet: Dissecting cis-versus-trans components for c-Src auto-phosphorylation. (A) WB of samples from a time-course auto-phosphorylation experiment with WT, Y419F and Y530F c-Src (3D-construct, 0.25-2.5 μM) in the presence of ATP (1 mM) and MgCl 2 (2 mM) for 0–60 min using the indicated c-Src phospho-Tyr 419 antibody. Total amount of protein was visualized by Coomassie staining. Diagram for the in-trans and cis-mechanism for auto-phosphorylation, lower inset. (B) WB of samples from a time-course phosphorylation assay (0-90 min) as in A, in the absence and in the presence of a c-Src KD K298M construct as an intact susbtrate surrogate using the indicated antibodies. Total amount of protein was visualized by Coomassie staining. Diagram for the enzyme (E) and susbtrate (S) acting kinases. lower inset. (C) WB of samples from a time-course experiment with c-Src KD WT in the presence of c-Src 3D-K298M constructs with and without Y419F and Y530F mutations as substrates using the indicated antibodies. Total c-Src protein was visualized by Coomassie staining. Diagram for the enzyme (E) and susbtrate (S) acting kinases. lower inset.

Article Snippet: Antibodies used were: phospho-Src Tyr419 (D49G4, CST #6943), Src (36D10, CST #2109) phospho-Src Tyr 530 (ThermoFisher 44-662G) and total phospho-Tyr (p-Tyr-100 CST #9411) were diluted at 1:10000-1:5000.

Techniques: Construct, Staining, Phosphorylation Assay

Journal: bioRxiv

Article Title: An allosteric switch between the activation loop and a c-terminal palindromic phospho-motif controls c-Src function

doi: 10.1101/2022.10.16.512342

Figure Lengend Snippet: (A) Schematic diagram of the functional domains and main auto-phosphorylation sites of c-Src. Different c-terminal sequence variants are depicted. (B) WB of samples from a time-course auto-phosphorylation experiment with c-Src WT, v-Src and 531X (3D-construct, 1 μM) in the presence of ATP (1 mM) and MgCl 2 (2 mM) for 0–60 min using the indicated antibodies. Total amount of protein was visualized by Coomassie staining. Phospho-tyrosine 216 quantification signal, data represent mean ± SEM of 3 experiments (n=3), * p < 0.05, one-way ANOVA test. (C) Enzyme kinetics and catalytic efficiency constant (k cat /K M , fold-change) for ATP using Src WT, v-Src and 531X (3D-construct, 1 μM final concentration) at fixed concentration (2-4 mg/ml) of Abl peptide. Data represent the mean ± SEM, of 3 experiments in duplicate (n=3). (D) Cartoon representation of the kinase-susbtrate (chain A and B) engagement of two c-Src molecules in the crystal structure. Left panel, close in view of the c-terminal aa sequence of c-Src containing the palindromic PQYQP motif at the interface between the two molecules showing residues coordinated by intra- and inter-molecular interactions. (E) WB of samples from a time-course phosphorylation experiment with c-Src WT, v-Src and 531X (3D-construct, 1 μM) in the presence of a c-Src KD K298M (3 μM) for 0–60 min using the indicated antibodies. Total amount of protein was visualized by Coomassie staining. (F) WB of samples from a time-course phosphorylation experiment with c-Src KD WT (1 μM), in the presence of susbtrate surrogates c-Src, v-Src and 531X K298M (3D-construct, 3 μM) for 0–60 min using the indicated antibodies. Total amount of protein was visualized by Coomassie staining. Phospho-tyrosine 419 and 530 quantification signal on the active kinase molecule, data represent mean ± SEM of 3 experiments (n=3), *** p = 0.0005, **** p = 0.0001, 2-way ANOVA test.

Article Snippet: Antibodies used were: phospho-Src Tyr419 (D49G4, CST #6943), Src (36D10, CST #2109) phospho-Src Tyr 530 (ThermoFisher 44-662G) and total phospho-Tyr (p-Tyr-100 CST #9411) were diluted at 1:10000-1:5000.

Techniques: Functional Assay, Sequencing, Construct, Staining, Concentration Assay

Journal: Nature Cancer

Article Title: A reversible SRC-relayed COX2 inflammatory program drives resistance to BRAF and EGFR inhibition in BRAF V600E colorectal tumors

doi: 10.1038/s43018-022-00508-5

Figure Lengend Snippet: a , b , Unsupervised hierarchical clustering of the phospho-catalytic activity signatures of WiDr cells treated with vemurafenib (VEM; n = 13 independent experiments) ± gefitinib (GEF; n = 5 independent experiments) or cetuximab (CET; n = 5 independent experiments) as compared to their untreated control counterparts ( n = 23 independent experiments). a , ATP consumption in cell extracts using 228 peptide sensors. b , Kinase signatures deconvoluted from the peptide phosphorylation profiles in a . Bar graphs next to the heatmaps show the P values (two-sided Student’s t test) for each of the peptides ( a ) or kinases ( b ) comparing all treated samples to controls. c , Volcano plot of the data in b displaying the change in kinase activity versus P value for each treatment arm (same as b : VEM, n = 13; VEM + GEF, n = 5; VEM + CET, n = 5; as compared to their untreated control counterparts ( n = 23), where n is the number of independent experiments). d , Bar graphs of the data in b representing the shift in activity of SRC, SFK, EGFR and HER family kinases when cells were treated with vemurafenib alone or in combination with gefitinib or cetuximab. Kinase activity is compared to that in untreated control cells, and data are displayed as the average ± standard error in nM of ATP. Same as in b , c : VEM, n = 13; VEM + GEF, n = 5; VEM + CET, n = 5; as compared to their untreated control counterparts ( n = 23), where n is the number of independent experiments. e , Representative IHC images showing staining intensity for active SFK (phosphorylated Y419 epitope in the SRC activation site) following treatment of a BRAF V600E CRC PDX model with vehicle control, dabrafenib (DAB) and/or trametinib (TRA) for 3 or 21 d. The color-coded bottom panel highlights differences in bin intensities from automated image analysis (see for details). IHC images and intensity quantifications are representative of n = 2 independent PDX tumors per treatment condition and n = 20 independent tissue areas per tumor and per condition. f , Quantification of IHC staining intensity for total and activated SFK in two PDX models treated for 3 or 21 d with dabrafenib ± trametinib versus vehicle control (two-sided Student’s t test, P < 1 × 10 –15 ). Using batch processing and automated analysis of IHC images, protein expression was measured at the single-cell level (that is, n ≥ 10,000 individual cancer cells per treatment condition and tumor). g , Proposed parallel mechanism of SRC activation in response to BRAF/MEK/EGFR therapies in BRAF V600E CRC. BRAF*, BRAF V600E .

Article Snippet: To detect SRC phosphorylated at Y419, phospho-Src (Y419) EGFR rabbit polyclonal antibody supplied by R&D Systems (AF2685) was used at a dilution of 1:50.

Techniques: Activity Assay, Control, Phospho-proteomics, Staining, Activation Assay, Immunohistochemistry, Expressing

Journal: Nature Cancer

Article Title: A reversible SRC-relayed COX2 inflammatory program drives resistance to BRAF and EGFR inhibition in BRAF V600E colorectal tumors

doi: 10.1038/s43018-022-00508-5

Figure Lengend Snippet: a , BRAF V600E CRC cell lines were treated with vemurafenib (VEM) for 7 to 8 hours. Vemurafenib was used at 1.75 uM in HT29, 2 uM in KM20, 0.15 uM in LIM2405, 2.25 uM in SNUC5, and 1.5 uM in WiDr (details of treatment conditions (concentration and time) are available in spreadsheets Supplementary Table and of the Supplementary Tables document). Cell lysates were assayed by western blot with the indicated antibodies. Upper panels: SFK activation is reflected by increased phosphorylation of the SRC activation site, Y419 (pY419). HSP90 is used as loading control. Bottom panel: reduction in ERK 1/2 phosphorylation as control of BRAF inhibition. Molecular weight/size markers are indicated on the right (kDa). The experiment was repeated ≥3 times with similar results. b , Representative IHC images showing total SRC staining intensity following treatment of a BRAF V600E CRC PDX model with vehicle control, dabrafenib (DAB) and/or trametinib (TRA) for 3 or 21 days. The color-coded bottom panel highlights differences in bin intensities resulting from automated image analysis (see Methods for details). A scale bar is provided (100 micrometers). As in main Fig. , IHC images and intensity quantifications are representative of n = 2 independent PDX tumors per treatment condition, and n = 20 independent tissue areas per tumor and per condition. c , Quantification of IHC staining intensity for total and activated SFK in two PDX models treated for 3 or 21 days with DAB ± TRA vs. vehicle control. As in main Fig. , we used batch processing and automated analysis of IHC images to quantify protein expression at the single cell level (that is, n ≥ 10,000 individual cancer cells per treatment condition and tumor). d , SRC staining score by IHC in untreated patient CRC tumor specimens with or without a BRAF V600E mutation, from primary (prim.) or metastatic (met.) sites.

Article Snippet: To detect SRC phosphorylated at Y419, phospho-Src (Y419) EGFR rabbit polyclonal antibody supplied by R&D Systems (AF2685) was used at a dilution of 1:50.

Techniques: Concentration Assay, Western Blot, Activation Assay, Phospho-proteomics, Control, Inhibition, Molecular Weight, Staining, Immunohistochemistry, Expressing, Mutagenesis

Journal: Nature Cancer

Article Title: A reversible SRC-relayed COX2 inflammatory program drives resistance to BRAF and EGFR inhibition in BRAF V600E colorectal tumors

doi: 10.1038/s43018-022-00508-5

Figure Lengend Snippet: a , BRAF V600E CRC cell lines treated with vemurafenib ± gefitinib were lysed and immunoblotted with the indicated antibodies. SFK activation is reflected by increased phosphorylation of the SRC activation site Y419 (pY419) and lack of phosphorylation of the inhibitory site Y530 (non-pY530). Active SRC can be deactivated by rephosphorylation of Y530 by CSK. HSP90 serves as a loading control. Molecular weight/size markers are indicated on the right (kDa). The experiment was repeated three times with similar results. b , Shift in vemurafenib sensitivity measured by cell viability assay (left) and calculation of the CI (right) upon treatment of BRAF V600E CRC or melanoma cell lines with vemurafenib + gefitinib ± a SRC inhibitor, dasatinib, for 3 d ( n = 4 independent experiments per cell line). c , Colony formation assays in which BRAF V600E CRC cells were treated with an increasing concentration of vemurafenib alone (control) or with a fixed dose of gefitinib ± dasatinib. Data are representative of n = 2 independent repeats. d , Treatment of cell line-derived xenograft mouse models with a vemurafenib progenitor, PLX4720 (PLX); dasatinib; saracatinib; and/or gefitinib for 21 d ( n = 7 mice per group). Plotted is the percent change in tumor volume relative to baseline (day 1). Data are displayed as the average for all mice in a specified treatment group ± standard error. e , Treatment of PDX models with vemurafenib ± gefitinib ± dasatinib for 21 d, with data plotted as in d ( n = 8 mice per group). All raw and relative tumor volumes and exact P values shown in d , e are available as Source Data; P values are from a two-sided Student’s t test. f , g , GLMs testing the association of change in tumor volume between treatment arms and vehicle over time shown in d , e . Effect size is measured as the GLM standard coefficient. A GLM was applied to each tumor model separately or combined. Results for cell line xenografts and PDXs are shown in f and g , respectively. GLM P values corrected for FDR are shown in g . NT, not tested. h , i , Comparison of the effect sizes and FDR-corrected P values of treatment arms with and without a SRC inhibitor. The same number of mice per group shown in d , e was used for the analyses in f – i (that is, n = 7 mice per treatment group for WiDr and KM20 cell line xenografts and n = 8 mice per treatment group for PDX models 1 and 2).

Article Snippet: To detect SRC phosphorylated at Y419, phospho-Src (Y419) EGFR rabbit polyclonal antibody supplied by R&D Systems (AF2685) was used at a dilution of 1:50.

Techniques: Activation Assay, Phospho-proteomics, Control, Molecular Weight, Viability Assay, Concentration Assay, Derivative Assay, Comparison

Journal: Nature Cancer

Article Title: A reversible SRC-relayed COX2 inflammatory program drives resistance to BRAF and EGFR inhibition in BRAF V600E colorectal tumors

doi: 10.1038/s43018-022-00508-5

Figure Lengend Snippet: a , Western blots to detect phospho-T202/Y204 ERK1/2 and total ERK1/2 in BRAF V600E CRC cell lines treated with vemurafenib (VEM) ± gefitinib (GEF) or dasatinib (DAS) collected after 8 h, 24 h, 48 h or 72 h. HSP90 is used as a loading control. The experiment was repeated 2 independent times with similar results. b , Quantification of western blots shown in panel ( a ). The bar plot (averages and standard deviations per treatment condition across cell lines) was overlaid with a dot plot displaying individual measurements per cell line and condition. Data are normalized to p-ERK levels after 8 h treatment with VEM alone. See table below for detailed values and color codes; n = 8 cell lines. c , Western blots to detect total and phospho-Y654 beta-catenin (CTNNB1) in BRAF V600E CRC cell lines treated with VEM, or GEF, or DAS, or combinations of VEM + GEF, or VEM + DAS, or VEM + GEF + DAS. The detection of phospho-Y419 and total SFK serves as a control for the effect of SFK-inhibition (with DAS). The experiment was repeated 3 independent times with similar results. In panels a , c , molecular weight/size markers are indicated on the right (kDa).

Article Snippet: To detect SRC phosphorylated at Y419, phospho-Src (Y419) EGFR rabbit polyclonal antibody supplied by R&D Systems (AF2685) was used at a dilution of 1:50.

Techniques: Western Blot, Control, Inhibition, Molecular Weight

Journal: Nature Cancer

Article Title: A reversible SRC-relayed COX2 inflammatory program drives resistance to BRAF and EGFR inhibition in BRAF V600E colorectal tumors

doi: 10.1038/s43018-022-00508-5

Figure Lengend Snippet: a , Levels of secreted PGE2 were measured by ELISA in the conditioned medium of BRAF V600E CRC cell lines treated with vemurafenib ± gefitinib. Data are displayed as the average PGE2 secretion in pg ml –1 per 100,000 cells ± s.d. ( n = 3 independent experiments per cell line). b , BRAF V600E CRC cell lines were treated with exogenous PGE2. Cell lysates were assayed by western blot as indicated. Y419 phosphorylation and lack of phosphorylation of Y530 (non-pY530) are used as readouts of SFK activation. HSP90 serves as a loading control. The experiment was repeated two independent times per cell line with similar results. c , Bar graphs representing fold change (log 2 scale) ± standard error for change in sensitivity to vemurafenib upon further treatment with PGE2 or untreated control in 3-day cell viability assays. Top, CI, Bliss model. Same methods as in Fig. ( n = 3 independent experiments per cell line). d , Western blots to detect pY654 of CTNNB1 in BRAF V600E CRC cell lines treated with exogenous PGE2. The experiment was repeated two independent times per cell line with similar results. e , Three BRAF V600E CRC cell lines engineered with a doxycycline-inducible constitutively active GNAS construct, iGNAS R201C , were treated with doxycycline. Cell lysates were assayed by western blot as indicated. The experiment was repeated three times with similar results. f , Bar graphs representing fold change (log 2 scale) ± standard error for change in sensitivity to vemurafenib or vemurafenib + gefitinib after iGNAS R201C induction in 3-day cell viability assays. Top, CI, as in c ( n = 3 independent experiments per cell line). g , GNAS was knocked out in BRAF V600E CRC cells using CRISPR (GNAS-KO). GNAS knockout was validated by western blot (top). GNAS-KO cells were treated with vemurafenib, and cell lysates were assayed by western blot with the indicated antibodies (bottom). The experiment was repeated ≥2 times with similar results. In b , d , e , g , molecular weight/size markers are indicated on the right (kDa). h , Bar graphs representing fold change (log 2 scale) ± standard error for change in sensitivity to vemurafenib or vemurafenib + gefitinib with GNAS knockout in 3-day cell viability assays. Top, CI, as in c ( n = 3 independent experiments per cell line). i , Representative IHC images showing COX2 staining intensity following treatment of a BRAF V600E CRC PDX model with vehicle control, dabrafenib and/or trametinib for 3 or 21 d (where n is the same as defined in Fig. ). The color-coded bottom panel highlights differences in bin intensities from automated image analysis (see for details). j , Quantification of COX2 staining intensity by IHC for two PDX models treated for 3 or 21 d with dabrafenib ± trametinib versus vehicle control (two-sided Student’s t test, P < 1 × 10 –15 ; n is the same as defined in Fig. ). k , Proposed mechanism of COX2–PGE2-mediated SRC-driven resistance to BRAF/MEK/EGFR therapies in BRAF V600E CRC.

Article Snippet: To detect SRC phosphorylated at Y419, phospho-Src (Y419) EGFR rabbit polyclonal antibody supplied by R&D Systems (AF2685) was used at a dilution of 1:50.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics, Activation Assay, Control, Construct, CRISPR, Knock-Out, Molecular Weight, Staining