pmcherry c1 vectors  (TaKaRa)

Journal: bioRxiv

Article Title: Membrane Curvature Activates Src kinase and Promotes Metastatic Cancer Cell Survival

doi: 10.64898/2026.03.03.709279

Figure Lengend Snippet: a, BFP channel of cytosolic condensates formed by 3×SH3 FBP17 –BFP, GFP–Nck1, N-WASP, and WIP, which fail to recruit Src(SH3–SH2) or Src(ΔSH4, Y530F). Scale bar, 10 µm. Related to . b, BFP channel of cytosolic condensates formed by 3×SH3 FBP17 –BFP, GFP–Nck1, N-WASP, and WIP, which recruit cortactin. Scale bar, 10 µm. Related to . c, BFP channel of cytosolic condensates formed by cortactin together with 3×SH3 FBP17 –BFP, GFP–Nck1, N-WASP, and WIP, which recruit Src(SH3–SH2) or Src(ΔSH4, Y530F). Scale bar, 10 µm. Related to . d, FRAP analysis of GFP–Nck1 and Src(ΔSH4, Y530F)–RFP in cytosolic condensates reveals rapid fluorescence recovery, indicating liquid-like dynamics. Nck1 recovers slightly faster than Src(ΔSH4, Y530F). Scale bars, 10 µm; 5 µm (montages). See also Supplementary Video 4. e, Sequences of the SH2- and SH3-binding motifs in adaptor proteins known to engage Src in a bipartite manner. Adaptor proteins include cortactin, Tks5, Tks4, AFAP1, FAK, Sam68, and p130Cas. f and g, Representative images of GFP-tagged adaptor proteins on nanobar substrates. Cortactin, Tks4, Tks5, and AFAP1 accumulate at nanobar ends, whereas FAK, Sam68, and p130Cas do not. Scale bars, 10 µm. h , Quantification of curvature enrichment (nanobar end-to-side ratios) for adaptor proteins shown in (e-g). Cortactin, Tks4, Tks5, and AFAP1 show significant enrichment, while FAK, Sam68, and p130Cas do not. n = 12 cells per condition, from two independent experiments; data are mean ± SD.

Article Snippet: These DNA fragments were used to replace the EGFP in pEGFP-N1 for mammalian expression, or inserted into pEGFP-N1, pEGFP-C1, pmCherry-N1 or pmCherry-C1 vector (Clontech) for the expression of GFP- or RFP-tagged constructs.

Techniques: Fluorescence, Binding Assay

Journal: bioRxiv

Article Title: Membrane Curvature Activates Src kinase and Promotes Metastatic Cancer Cell Survival

doi: 10.64898/2026.03.03.709279

Figure Lengend Snippet: a, Schematic illustrating curvature-induced Src activation: condensates formed at membrane curvature enrich open-conformation Src, promoting local trans-autophosphorylation at Y419. b, Ratiometric imaging of Src(pY419)/Src–GFP and Src(pY419)/Src(Y530F)–GFP reveals enhanced Y419 phosphorylation at nanopillars. Scale bar, 10 µm. c, Quantification confirms that Src autophosphorylation (pY419) is significantly elevated at nanopillars compared to flat regions or focal adhesions. n = 12 (Src–GFP) and 15 (Src(Y530F)–GFP) cells, from two independent experiments; data are mean ± SD; p values from one-way ANOVA with Dunnett’s T3 multiple comparisons. d, Immunostaining for total Src, open conformation Src(Y530), and active Src(pY419) shows different degrees of enrichment at nanopillars. Scale bar, 10 µm. e, Quantification reveals highest curvature enrichment for Src(pY419), intermediate for Src(Y530), and minimal for total Src. n = 22, 19, 21 cells from two independent experiments; mean ± SD; p values from one-way ANOVA with Dunnett’s T3 multiple comparisons. f, At nanopillars, Src(Y530F)–RFP colocalizes with N-WASP/WIP/GFP–Nck1 condensates, while Csk–RFP (a negative regulator of Src) is excluded. Scale bar, 10 µm. g, Schematic illustrating PI3K/Akt signaling downstream of curvature-induced Src activation. h, PI3K regulatory subunit alpha (p85α–GFP) accumulates at nanopillars, colocalizing with Src(pY419). Scale bar, 10 µm. i, Live-cell imaging showing Akt–PH–GFP, a PI(3,4,5)P3 sensor, enriched at nanopillars together with Src(Y530F)–RFP. Scale bar, 10 µm. j, Treatment with Src inhibitor dasatinib abolishes p85α–GFP and Src(pY419) enrichment at nanopillars. Scale bar, 10 µm. k, Dasatinib treatment eliminates p85α–GFP enrichment at nanopillars, while Src(Y530F)–RFP enrichment remains, indicating that curvature localization of Src(Y530F) does not rely on its kinase activity. Scale bar, 10 µm. l, PI3K inhibition (ZSTK474) does not affect Src(pY419) or p85α–GFP enrichment at nanopillars. Scale bar, 10 µm. m, Enrichment of Akt–PH–GFP at nanopillars is abolished by either Src or PI3K inhibition, indicating that both kinases are required for Akt signaling at curvature. Scale bar, 10 µm. n, Quantification of curvature enrichment for p85α-GFP, Src(pY419), and Akt–PH–GFP under the indicated inhibitor treatments. n = 19, 18, 21, 17, and 16 cells (p85α-GFP co-stained with Src(pY419)); 20, 15, 17, 19, and 19 cells (Akt–PH–GFP), from two independent experiments; data are mean ± SD; p -values from Kruskal-Wallis tests with Dunn’s multiple comparisons.

Article Snippet: These DNA fragments were used to replace the EGFP in pEGFP-N1 for mammalian expression, or inserted into pEGFP-N1, pEGFP-C1, pmCherry-N1 or pmCherry-C1 vector (Clontech) for the expression of GFP- or RFP-tagged constructs.

Techniques: Activation Assay, Membrane, Imaging, Phospho-proteomics, Immunostaining, Live Cell Imaging, Activity Assay, Inhibition, Staining

Journal: bioRxiv

Article Title: Membrane Curvature Activates Src kinase and Promotes Metastatic Cancer Cell Survival

doi: 10.64898/2026.03.03.709279

Figure Lengend Snippet: a , GFP-tagged nSH2–cSH2 fragment of p85α accumulates at nanopillars and colocalizes with Src(pY419). Scale bar, 10 µm. b, Immunostaining of p85α(pY467) showing its strong curvature-dependent enrichment at nanopillars, colocalizing with Src(pY419). Scale bar, 10 µm. c , Treatment with Src inhibitor saracatinib abolishes the enrichment of p85α–GFP and Src(pY419) at nanopillars, similar to the effect of dasatinib. Scale bar, 10 µm. d, Despite saracatinib treatment, Src(Y530F)–RFP remains enriched at nanopillars, while p85α–GFP enrichment is abolished, indicating that Src kinase activity is necessary for PI3K but not Src recruitment to curvature. Scale bar, 10 µm. e, Immunostaining confirms that both dasatinib and saracatinib eliminate nanopillar enrichment of Src(pY419) and p85α(pY467). Scale bar, 10 µm.

Article Snippet: These DNA fragments were used to replace the EGFP in pEGFP-N1 for mammalian expression, or inserted into pEGFP-N1, pEGFP-C1, pmCherry-N1 or pmCherry-C1 vector (Clontech) for the expression of GFP- or RFP-tagged constructs.

Techniques: Immunostaining, Activity Assay