AF4607 Search Results


pstat3 y705  (R&D Systems)
93
R&D Systems pstat3 y705
misPLA analyses of phosphorylation states and protein-protein interactions in SK-BR cells with or without EGF stimulation. A) Phosphorylation targets: SK-BR cells were analyzed for phosphorylation of STAT5a (pSTAT5a), STAT3 <t>(pSTAT3),</t> AKT (pAKT), ERK (pERK), and EGFR (pEGFR), under unstimulated and EGF-stimulated conditions. All targets were visualized three at a time in sequential detection cycles and are shown simultaneously (upper panels, “All targets”) and subsequently as individual channels. PLA signals (red, cyan, green, purple) reflect activated protein states detected via dual-recognition proximity ligation. DAPI (blue) labels nuclei. Scale bars = 50 µm. B) Protein-protein interactions: Visualization of the following protein-protein interactions investigated by misPLA in unstimulated and EGF-stimulated SK-BR cells: MEK1–ERK2, GRB2–MEK1, EGFR–GRB2, STAT3–STAT5a, JAK1–JAK3, JAK1–PI3K, JAK2–STAT5a, JAK1–STAT3, and JAK2–JAK3. Upper panels (“All targets”) represent simultaneous visualization of all targets, imaged three at a time in sequential detection cycles, followed by separated signals per interaction. Scale bars = 50 µm.
Pstat3 Y705, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/AF4607/Human+Phospho-STAT3+(Y705)+Antibody/bio_rxiv__2025__07__11__662357-213-37-38
Average 93 stars, based on 1 article reviews
pstat3 y705 - by Bioz Stars, 2026-10
93/100 stars
  Buy from Supplier
anti pstat3  (R&D Systems)
94
R&D Systems anti pstat3
misPLA analyses of phosphorylation states and protein-protein interactions in SK-BR cells with or without EGF stimulation. A) Phosphorylation targets: SK-BR cells were analyzed for phosphorylation of STAT5a (pSTAT5a), STAT3 <t>(pSTAT3),</t> AKT (pAKT), ERK (pERK), and EGFR (pEGFR), under unstimulated and EGF-stimulated conditions. All targets were visualized three at a time in sequential detection cycles and are shown simultaneously (upper panels, “All targets”) and subsequently as individual channels. PLA signals (red, cyan, green, purple) reflect activated protein states detected via dual-recognition proximity ligation. DAPI (blue) labels nuclei. Scale bars = 50 µm. B) Protein-protein interactions: Visualization of the following protein-protein interactions investigated by misPLA in unstimulated and EGF-stimulated SK-BR cells: MEK1–ERK2, GRB2–MEK1, EGFR–GRB2, STAT3–STAT5a, JAK1–JAK3, JAK1–PI3K, JAK2–STAT5a, JAK1–STAT3, and JAK2–JAK3. Upper panels (“All targets”) represent simultaneous visualization of all targets, imaged three at a time in sequential detection cycles, followed by separated signals per interaction. Scale bars = 50 µm.
Anti Pstat3, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/AF4607/Human+Phospho-STAT3+(Y705)+Antibody/pmc06302113-150-31-33
Average 94 stars, based on 1 article reviews
anti pstat3 - by Bioz Stars, 2026-10
94/100 stars
  Buy from Supplier

Image Search Results


misPLA analyses of phosphorylation states and protein-protein interactions in SK-BR cells with or without EGF stimulation. A) Phosphorylation targets: SK-BR cells were analyzed for phosphorylation of STAT5a (pSTAT5a), STAT3 (pSTAT3), AKT (pAKT), ERK (pERK), and EGFR (pEGFR), under unstimulated and EGF-stimulated conditions. All targets were visualized three at a time in sequential detection cycles and are shown simultaneously (upper panels, “All targets”) and subsequently as individual channels. PLA signals (red, cyan, green, purple) reflect activated protein states detected via dual-recognition proximity ligation. DAPI (blue) labels nuclei. Scale bars = 50 µm. B) Protein-protein interactions: Visualization of the following protein-protein interactions investigated by misPLA in unstimulated and EGF-stimulated SK-BR cells: MEK1–ERK2, GRB2–MEK1, EGFR–GRB2, STAT3–STAT5a, JAK1–JAK3, JAK1–PI3K, JAK2–STAT5a, JAK1–STAT3, and JAK2–JAK3. Upper panels (“All targets”) represent simultaneous visualization of all targets, imaged three at a time in sequential detection cycles, followed by separated signals per interaction. Scale bars = 50 µm.

Journal: bioRxiv

Article Title: Spatial mapping of proteins and their activity states in cancer models by multiplex in situ PLA

doi: 10.1101/2025.07.11.662357

Figure Lengend Snippet: misPLA analyses of phosphorylation states and protein-protein interactions in SK-BR cells with or without EGF stimulation. A) Phosphorylation targets: SK-BR cells were analyzed for phosphorylation of STAT5a (pSTAT5a), STAT3 (pSTAT3), AKT (pAKT), ERK (pERK), and EGFR (pEGFR), under unstimulated and EGF-stimulated conditions. All targets were visualized three at a time in sequential detection cycles and are shown simultaneously (upper panels, “All targets”) and subsequently as individual channels. PLA signals (red, cyan, green, purple) reflect activated protein states detected via dual-recognition proximity ligation. DAPI (blue) labels nuclei. Scale bars = 50 µm. B) Protein-protein interactions: Visualization of the following protein-protein interactions investigated by misPLA in unstimulated and EGF-stimulated SK-BR cells: MEK1–ERK2, GRB2–MEK1, EGFR–GRB2, STAT3–STAT5a, JAK1–JAK3, JAK1–PI3K, JAK2–STAT5a, JAK1–STAT3, and JAK2–JAK3. Upper panels (“All targets”) represent simultaneous visualization of all targets, imaged three at a time in sequential detection cycles, followed by separated signals per interaction. Scale bars = 50 µm.

Article Snippet: The following primary antibodies were used for Western blotting: JAK1 (ProteinTech, 66466-1-Ig), STAT3 (ProteinTech, 60199-1-Ig; Abcam, ab171359), MEK1 (Abcam, ab239802), EGFR (Abcam, ab271834), AKT2 (Thermo Scientific, PA5-85518), ERK2 (Thermo Fisher, PA5-29636), phospho-PI3K p85/p55 (Cell Signaling Technology, 4228S), pSTAT3-Y705 (R&D Systems, AF4607), Grb2 (R&D Systems, mab38461), GAPDH (CST, 14C10), and Vinculin (CST, E1E9V), used at either 1:1000 or 1:2000 dilution.

Techniques: Phospho-proteomics, Protein-Protein interactions, Ligation

In A) Sk-BR-3 human HER2-positive breast-carcinoma cells were stimulated with epidermal growth factor (EGF; 50 ng/ml) for 60 min, or left either serum-starved (Starvation control) or in complete-media control conditions; whole-cell lysate from K562 cells was loaded as a positive control in the right-most lane. Immunoblots were probed sequentially for STAT3 (≈70 kDa; Abcam, ab171359, 1:2 000), STAT5A (≈80 kDa; Abcam, ab213219, 1:1 000), ERK2 (≈42 kDa; Thermo Fisher, PA5-29636, 1:1 000), MEK1 (≈45 kDa; Abcam, ab239802, 1:1 000), AKT2 (≈55 kDa; Thermo Fisher, PA5-85518, 1:1 000), phospho-STAT3-Tyr705 (pSTAT3, ≈90–115 kDa; R&D, AF4607, 1:2 000) and pan-AKT (≈60 kDa; CST, #4691, 1:1 000). Membranes were finally reprobed for GAPDH (≈37 kDa; CST, #14C10, 1:1 000) and vinculin (≈120 kDa; CST, #E1E9V, 1:1 000) as loading controls. Molecular-weight markers are indicated on the left. For all panels, proteins were resolved on Bis-Tris SDS–PAGE gels (4–12 % or 10 % as specified in Methods) using MES or MOPS running buffer, transferred to PVDF, and detected by near-infrared fluorescence on a LI-COR Odyssey® imager. In B) estimated relative band intensities of key signaling proteins in Sk-BR-3 and K562 cells are shown. Fluorescence-based Western blot images (Supp (A)) were used to visually estimate band intensities of STAT3, phospho-STAT3 (Tyr705), STAT5A, ERK2, MEK1, AKT2, and pan-AKT in Sk-BR-3 cells under three conditions: serum starvation, complete media, and 60 min EGF treatment, and in K562 cells as a positive control. Band intensities were approximated by visual comparison of signal brightness and normalized to the corresponding housekeeping protein (GAPDH or vinculin) within each lane. Normalized values were plotted as grouped bar graphs to highlight relative abundance patterns across conditions. This semi-quantitative representation supports qualitative comparison and was not derived from raw densitometry. validates the antibody specificity and establishes the baseline expression of PLA target proteins in Sk-BR-3 and K562 cells. Panel A displays the Western blot images of Sk-BR-3 cells subjected to three conditions: serum starvation, complete media (control), and EGF stimulation for 60 minutes. K562 cell lysate is included as a positive control. Panel B presents the quantified band intensities, normalised to the housekeeping proteins GAPDH or vinculin and expressed as percentages. In Sk-BR-3 cells, total STAT3, ERK2, MEK1, AKT2, and pan-AKT were consistently detected across all conditions. STAT3 levels remained stable, while phosphorylation at Tyr705 (pSTAT3) dropped to undetectable levels under serum starvation, rose slightly under media control, and reached higher levels after 60 min of EGF treatment. This trend suggests growth-factor-responsive STAT3 activation despite unchanged total STAT3 levels. MEK1 and AKT2 expression decreased mildly in media control compared to both EGF and starvation conditions, while ERK2 and pan-AKT remained constant. STAT5A was not detected in any Sk-BR3 condition. In contrast, the K562 lysate showed robust STAT5A expression, confirming its hematopoietic lineage specificity. STAT3 was present at lower levels relative to Sk-BR-3 media control, while ERK2 and pan-AKT were comparable between both cell types. MEK1 and AKT2 appeared at slightly lower levels in K562 cells. GAPDH and vinculin levels were consistent across all samples, confirming equal loading and transfer quality. Together, these results confirm the specificity of each antibody, the absence of non-specific binding, and the suitability of both Sk-BR-3 and K562 cells as models for downstream proximity ligation assays aimed at monitoring dynamic signalling interactions.

Journal: bioRxiv

Article Title: Spatial mapping of proteins and their activity states in cancer models by multiplex in situ PLA

doi: 10.1101/2025.07.11.662357

Figure Lengend Snippet: In A) Sk-BR-3 human HER2-positive breast-carcinoma cells were stimulated with epidermal growth factor (EGF; 50 ng/ml) for 60 min, or left either serum-starved (Starvation control) or in complete-media control conditions; whole-cell lysate from K562 cells was loaded as a positive control in the right-most lane. Immunoblots were probed sequentially for STAT3 (≈70 kDa; Abcam, ab171359, 1:2 000), STAT5A (≈80 kDa; Abcam, ab213219, 1:1 000), ERK2 (≈42 kDa; Thermo Fisher, PA5-29636, 1:1 000), MEK1 (≈45 kDa; Abcam, ab239802, 1:1 000), AKT2 (≈55 kDa; Thermo Fisher, PA5-85518, 1:1 000), phospho-STAT3-Tyr705 (pSTAT3, ≈90–115 kDa; R&D, AF4607, 1:2 000) and pan-AKT (≈60 kDa; CST, #4691, 1:1 000). Membranes were finally reprobed for GAPDH (≈37 kDa; CST, #14C10, 1:1 000) and vinculin (≈120 kDa; CST, #E1E9V, 1:1 000) as loading controls. Molecular-weight markers are indicated on the left. For all panels, proteins were resolved on Bis-Tris SDS–PAGE gels (4–12 % or 10 % as specified in Methods) using MES or MOPS running buffer, transferred to PVDF, and detected by near-infrared fluorescence on a LI-COR Odyssey® imager. In B) estimated relative band intensities of key signaling proteins in Sk-BR-3 and K562 cells are shown. Fluorescence-based Western blot images (Supp (A)) were used to visually estimate band intensities of STAT3, phospho-STAT3 (Tyr705), STAT5A, ERK2, MEK1, AKT2, and pan-AKT in Sk-BR-3 cells under three conditions: serum starvation, complete media, and 60 min EGF treatment, and in K562 cells as a positive control. Band intensities were approximated by visual comparison of signal brightness and normalized to the corresponding housekeeping protein (GAPDH or vinculin) within each lane. Normalized values were plotted as grouped bar graphs to highlight relative abundance patterns across conditions. This semi-quantitative representation supports qualitative comparison and was not derived from raw densitometry. validates the antibody specificity and establishes the baseline expression of PLA target proteins in Sk-BR-3 and K562 cells. Panel A displays the Western blot images of Sk-BR-3 cells subjected to three conditions: serum starvation, complete media (control), and EGF stimulation for 60 minutes. K562 cell lysate is included as a positive control. Panel B presents the quantified band intensities, normalised to the housekeeping proteins GAPDH or vinculin and expressed as percentages. In Sk-BR-3 cells, total STAT3, ERK2, MEK1, AKT2, and pan-AKT were consistently detected across all conditions. STAT3 levels remained stable, while phosphorylation at Tyr705 (pSTAT3) dropped to undetectable levels under serum starvation, rose slightly under media control, and reached higher levels after 60 min of EGF treatment. This trend suggests growth-factor-responsive STAT3 activation despite unchanged total STAT3 levels. MEK1 and AKT2 expression decreased mildly in media control compared to both EGF and starvation conditions, while ERK2 and pan-AKT remained constant. STAT5A was not detected in any Sk-BR3 condition. In contrast, the K562 lysate showed robust STAT5A expression, confirming its hematopoietic lineage specificity. STAT3 was present at lower levels relative to Sk-BR-3 media control, while ERK2 and pan-AKT were comparable between both cell types. MEK1 and AKT2 appeared at slightly lower levels in K562 cells. GAPDH and vinculin levels were consistent across all samples, confirming equal loading and transfer quality. Together, these results confirm the specificity of each antibody, the absence of non-specific binding, and the suitability of both Sk-BR-3 and K562 cells as models for downstream proximity ligation assays aimed at monitoring dynamic signalling interactions.

Article Snippet: The following primary antibodies were used for Western blotting: JAK1 (ProteinTech, 66466-1-Ig), STAT3 (ProteinTech, 60199-1-Ig; Abcam, ab171359), MEK1 (Abcam, ab239802), EGFR (Abcam, ab271834), AKT2 (Thermo Scientific, PA5-85518), ERK2 (Thermo Fisher, PA5-29636), phospho-PI3K p85/p55 (Cell Signaling Technology, 4228S), pSTAT3-Y705 (R&D Systems, AF4607), Grb2 (R&D Systems, mab38461), GAPDH (CST, 14C10), and Vinculin (CST, E1E9V), used at either 1:1000 or 1:2000 dilution.

Techniques: Control, Positive Control, Western Blot, Molecular Weight, SDS Page, Fluorescence, Comparison, Derivative Assay, Expressing, Phospho-proteomics, Activation Assay, Binding Assay, Ligation

Analysis of primary blood cells from two patients diagnosed with CML, targeting molecular pathways known to be up-regulated in CML. The experiment used a slightly different oligonucleotide design compared to other experiments reported herein, but with similar performance ( .) A, E) Quantitative analysis of numbers of signals per cell revealed striking differences among individual blood cells. Top and bottom rows represent data for two different CML patients. Visualization cycle 1: phosphoPI3K-AKT1 in Cy3 (red), phosphoAKT1-AKT3 in Cy5 (green), AKT(pan)-AKT2 in FITC (yellow). Cycle 2: JAK2-JAK3 in Cy3 (blue), phosphorylated GRB2 in Cy5 (orange), MEK1-ERK in FITC (purple). Cycle 3: STAT3-phosphoSTAT3 in Cy5 (cyan), STAT3-phosphoSTAT3 in FITC (magenta) and STAT3-STAT5 in Cy3 (lime). The same color coding was used throughout all panels in the figure. B, F) Scatterplots of pairs of detection reactions, serving to visualize correlations across detection pairs (colors as in A, B)). C, G) Visualization of a zoomed-in view of detection reactions per cell. Outline of nuclei are shown in red. D, H) Cell to cell heterogeneity is visualized by overlaying individual detection reactions on the cells - here showing zoomed in region with D) phosphorylated GRB2 (in orange) and (h) STAT3-phosphoSTAT3 (in magenta). Cell nuclei are outlined in red. The raw image data from the full sample, together with detections are available for interactive viewing at https://ulflandegren2025.serve.scilifelab.se .

Journal: bioRxiv

Article Title: Spatial mapping of proteins and their activity states in cancer models by multiplex in situ PLA

doi: 10.1101/2025.07.11.662357

Figure Lengend Snippet: Analysis of primary blood cells from two patients diagnosed with CML, targeting molecular pathways known to be up-regulated in CML. The experiment used a slightly different oligonucleotide design compared to other experiments reported herein, but with similar performance ( .) A, E) Quantitative analysis of numbers of signals per cell revealed striking differences among individual blood cells. Top and bottom rows represent data for two different CML patients. Visualization cycle 1: phosphoPI3K-AKT1 in Cy3 (red), phosphoAKT1-AKT3 in Cy5 (green), AKT(pan)-AKT2 in FITC (yellow). Cycle 2: JAK2-JAK3 in Cy3 (blue), phosphorylated GRB2 in Cy5 (orange), MEK1-ERK in FITC (purple). Cycle 3: STAT3-phosphoSTAT3 in Cy5 (cyan), STAT3-phosphoSTAT3 in FITC (magenta) and STAT3-STAT5 in Cy3 (lime). The same color coding was used throughout all panels in the figure. B, F) Scatterplots of pairs of detection reactions, serving to visualize correlations across detection pairs (colors as in A, B)). C, G) Visualization of a zoomed-in view of detection reactions per cell. Outline of nuclei are shown in red. D, H) Cell to cell heterogeneity is visualized by overlaying individual detection reactions on the cells - here showing zoomed in region with D) phosphorylated GRB2 (in orange) and (h) STAT3-phosphoSTAT3 (in magenta). Cell nuclei are outlined in red. The raw image data from the full sample, together with detections are available for interactive viewing at https://ulflandegren2025.serve.scilifelab.se .

Article Snippet: The following primary antibodies were used for Western blotting: JAK1 (ProteinTech, 66466-1-Ig), STAT3 (ProteinTech, 60199-1-Ig; Abcam, ab171359), MEK1 (Abcam, ab239802), EGFR (Abcam, ab271834), AKT2 (Thermo Scientific, PA5-85518), ERK2 (Thermo Fisher, PA5-29636), phospho-PI3K p85/p55 (Cell Signaling Technology, 4228S), pSTAT3-Y705 (R&D Systems, AF4607), Grb2 (R&D Systems, mab38461), GAPDH (CST, 14C10), and Vinculin (CST, E1E9V), used at either 1:1000 or 1:2000 dilution.

Techniques: