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: 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

Journal: Discover oncology

Article Title: Hepatoma cell-derived exosomal SNORD52 mediates M2 macrophage polarization by activating the JAK2/STAT6 pathway.

doi: 10.1007/s12672-024-01700-y

Figure Lengend Snippet: Fig. 3 SNORD52 mediates M2 macrophage polariza- tion and activates JAK2/ STAT6 pathway. A Following transfection of OE-SNORD52/ OE-NC in THP-1 and U937 for 48 h, the mRNA expression of SNORD52 was detected by qRT-PCR. ****P < 0.0001 vs. OE-NC. B Following trans- fection of OE-SNORD52/ OE-NC in THP-1 and U937 for 48 h, the protein levels of Arginase-1 and CD163 were determined through Western blotting. *P < 0.05, **P < 0.01, ***P < 0.001 vs. OE-NC. C The ratio of CD206-positive or CD163-positive cells following transfection of OE-SNORD52/ OE-NC was analyzed by flow cytometry. ***P < 0.001 vs. OE-NC. D Following transfec- tion of OE-SNORD52/OE-NC in THP-1 and U937 for 48 h, the levels of JAK2/STAT6 pathway-related proteins were determined through Western blotting. ***P < 0.001 vs. OE-NC. E The IL-4 and IL-13 treated THP-1/U937 mac- rophages were co-incubated with SNORD52 ASO-1/-2 or ASO-NC for 48 h, and then the mRNA expression of SNORD52 was detected by qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001 vs. ASO-NC. F The IL-4 and IL-13 treated THP-1/U937 mac- rophages were co-incubated with SNORD52 ASO-1/-2 or ASO-NC for 48 h, and then the protein levels of Arginase-1 and CD163 were determined through Western blotting. ***P < 0.001 vs. ASO-NC. G The ratio of CD206-positive or CD163-positive cells following transfection of SNORD52- ASO-1/-2 or ASO-NC was analyzed by flow cytometry. ***P < 0.001 vs. ASO-NC. (H) The IL-4 and IL-13 treated THP-1/ U937 macrophages were co-incubated with SNORD52 ASO-1/-2 or ASO-NC for 48 h, and then the levels of JAK2/STAT6 pathway-related proteins were determined through Western blotting. ***P < 0.001 vs. ASO-NC. ns: no significance

Article Snippet: Meanwhile, the primary antibodies Arginase-1, CD163, and STAT6 used in Western blotting were also purchased from Proteintech, while other primary antibodies including JAK2, p-JAK2, p-STAT6, exosome-positive markers (CD81 and TSG101), and the corresponding HRP-conjugated secondary antibodies were available from Abcam (Cambridge, UK).

Techniques: Transfection, Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Incubation

Journal: Discover oncology

Article Title: Hepatoma cell-derived exosomal SNORD52 mediates M2 macrophage polarization by activating the JAK2/STAT6 pathway.

doi: 10.1007/s12672-024-01700-y

Figure Lengend Snippet: Fig. 5 Huh7 cells-derived exosomal SNORD52 mediates M2 macrophage polarization through activating JAK2/STAT6 pathway. A Following the establishment of co-culture model, AG-490 (a specific JAK2 inhibitor) was added and the levels of JAK2/STAT6 pathway-related pro- teins in receptor THP-1 macrophages were measured by Western blotting. B Following the establishment of co-culture model, AG-490 (a specific JAK2 inhibitor) was added and the ratio of CD206-positive or CD163-positive THP-1 macrophages was analyzed by flow cytometry. **P < 0.01, ***P < 0.001. ns: no significance

Article Snippet: Meanwhile, the primary antibodies Arginase-1, CD163, and STAT6 used in Western blotting were also purchased from Proteintech, while other primary antibodies including JAK2, p-JAK2, p-STAT6, exosome-positive markers (CD81 and TSG101), and the corresponding HRP-conjugated secondary antibodies were available from Abcam (Cambridge, UK).

Techniques: Derivative Assay, Co-Culture Assay, Western Blot, Flow Cytometry

Journal: Journal of molecular histology

Article Title: tsRNA-3043a intensifies apoptosis and senescence of ovarian granulosa cells to drive premature ovarian failure by targeting FLT1.

doi: 10.1007/s10735-024-10256-8

Figure Lengend Snippet: Fig. 10 Diagram of the hypothesis mechanism of tsRNA-3043a facilitates POF by inhibiting FLT1

Article Snippet: The sections were blocked with 5% BSA and incubated with the anti-FLT1 antibody (1:200, 13687-1-AP, Proteintech) overnight at 4°C.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Systemic lupus erythematosus immune complexes increase the expression of SLAM family members CD319 (CRACC) and CD229 (LY-9) on plasmacytoid dendritic cells and CD319 on CD56(dim) NK cells.

doi: 10.4049/jimmunol.1301022

Figure Lengend Snippet: FIGURE 5. pDCs lack the activating adaptor molecules but express inhibitory adaptor molecules of SLAM receptors. (A) IFN-a production by RNA-IC–stimulated pDCs after cross-linking of CD319, CD229, or BDCA-2 by plate-bound mAbs. IFN-a levels were determined in 20-h cell culture supernatants by an immunoassay and normalized to the IFN-a production in cultures without plate-bound mAb. The dashed line indicates 100%. Data (mean + SD) are from three donors from two independent experiments. Expression of EAT2 and SAP (B) and SHIP-1, SHP-1, SHP- 2, and CSK (C) in cell lysates from isolated pDCs or NK cells from two healthy donors was determined by Western blot. Expression of b-actin was used as control.

Article Snippet: Rabbit polyclonal Abs to Ewing’s sarcoma-activated transcript 2 (EAT2), SHIP-1, SHP-2, CSK (Proteintech Group, Chicago, IL), and a rabbit mAb to SHP-1 (EPR5519; Epitomics, Burlingame, CA), followed by HRP-conjugated goat anti-rabbit IgG (H+L) (Invitrogen), were used to detect the indicated signaling molecules.

Techniques: Cell Culture, Expressing, Isolation, Western Blot, Control

Journal: International Journal of Nanomedicine

Article Title: Intravenous Injection of Na Ions Aggravates Ang II-Induced Hypertension-Related Vascular Endothelial Injury by Increasing Transmembrane Osmotic Pressure

doi: 10.2147/IJN.S435144

Figure Lengend Snippet: Role of mechanosensitive protein Abl2 in inflammation to regulate transmembrane OP in Ang II–induced HUVECs treated with sodium ion injection. HUVECs were incubated with Ang II and 0.9% NaCl, Compound NaCl, or 5% NaHCO 3 injection for 24 h (mixed with plasma-mimicking medium at 1:9). ( A ) Representative images of cells immunostained with Abl2 and actin (scale bar: 20 μm). ( B ) Pearson curve was used to show colocalization of Abl2 protein and actin between points A and B in each group in . ( C ) Expression of Abl2 in HUVECs transfected with Abl2 siRNA. Hypertensive HUVECs transfected with or without Abl2 siRNA were incubated with 0.9% NaCl, Compound NaCl, or 5% NaHCO 3 injection. ( D ) Representative images of cells immunostained with ASC (FITC) and NLRP3 (TRITC) (scale bar: 20 μm). ( E ) Quantitative statistics of fluorescence intensity of ASC and NLRP3. ( F ) Immunofluorescence staining of ROS in HUVECs (scale bar = 40 μm). ( G ) Quantitative statistics for fluorescence intensity of ROS. ( H ) Images obtained at 15-min time lapse in FRET analysis (calibration bar: 0.1–1.5; scale bar: 10 µm). ( I ) Standardized CFP/FRET ratio of vimentin within 15 min. ( J ) Ratio of transendothelial electrical resistance (TEER) values of HUVECs at 12 h and 0 h. NC, normal; Ang II, Angiotensin II. Data are expressed as mean ± SD (n = 3). * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: Abl2 antibodies were from Proteintech (Chicago, USA).

Techniques: Injection, Incubation, Clinical Proteomics, Expressing, Transfection, Fluorescence, Immunofluorescence, Staining