Journal: eLife

Article Title: Decoding protein phosphorylation during oocyte meiotic divisions using phosphoproteomics

doi: 10.7554/eLife.104255

Figure Lengend Snippet: ( A ) Comparison of phosphorylation changes during meiotic divisions in Xenopus (our dataset) and mouse oocytes . Log2 fold changes in phosphorylation between prophase (PRO) and metaphase II are plotted. The Pearson correlation ( r =0.39, p<0.0001) was calculated. ( B ) Selected examples of conserved phosphorylation sites between mouse and Xenopus . The kinase that was experimentally identified to phosphorylate the specific site is displayed together with the supporting reference. ( C ) Oocytes were microinjected with a specific inhibitor of Cdk1, Cip1. After overnight incubation, meiotic maturation was induced by progesterone. Oocytes were collected at different times after progesterone treatment. Karyopherin was used as a loading control. The phosphorylation of Fak1 at S913, Erk2 at T188, Plk1 at T201, and Akts1 at S208 was evaluated by western blot. Figure 10—source data 1. Original files of the full raw uncropped blots of . Figure 10—source data 2. Original western blots indicating the relevant bands used in .

Article Snippet: The following antibodies were used: Karyopherin (1:2000, goat, Santa Cruz sc-1863), pT188-pY190-Erk2 (1:2000, mouse, Cell Signaling 9106), pT201-Plk1 (1:1000, Abcam Ab39068), pS910-FAK (1:5000, rabbit, Invitrogen 4459 G) and pS183-Akts1/PRAS40 (1:5000, rabbit, Cell Signaling 5936).

Techniques: Comparison, Phospho-proteomics, Incubation, Control, Western Blot

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

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

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 mapping of signaling interactions in a lymph-node Hodgkin lymphoma, mixed cellularity (right neck); Hodgkin lymphoma, lymphocyte-depleted (neck); Hodgkin lymphoma, lymphocyte-predominant (left neck); Hodgkin lymphoma, mixed cellularity (left neck); and thymoma type B3 (mediastinum). Top row (visualization cycle 1) displays MEK1–ERK2 (FITC), EGFR–GRB2 (Cy5) and GRB2–MEK1 (Cy3N) together with DAPI. Middle row (cycle 2) shows STAT3–STAT5a (FITC), JAK1–JAK3 (Cy5) and JAK1–PI3Kp85 (Cy3N). Bottom row (cycle 3) presents JAK2– STAT5a (FITC), JAK2–JAK3 (Cy5) and JAK1–STAT3 (Cy3N). All nine pairs of antibody-oligonucleotide conjugates were applied and then amplified in a single incubation. The RCA products were revealed using detection oligonucleotides conjugated with three fluorophores in three visualization cycles. A standard three-channel fluorescence microscope was used with identical settings for all three fluorophores. 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: Amplification, Incubation, Fluorescence, Microscopy