Journal: bioRxiv

Article Title: NDR1/2 kinases regulate cell polarization and cell motility through Cdc42 GTPase and Pard3 signaling in mammalian cells

doi: 10.1101/2025.10.30.685405

Figure Lengend Snippet: A-C Fibroblasts were fixed 10 h after wound scratch and subjected to immunofluorescence analysis. Pard3 was visualized with Alexa Fluor 647 (A). Junctional organization was quantified by lacunarity (B) and average puncta size (C)., ****P < 0. 0001, ***P < 0.001, ** P < 0.01, One-Way ANOVA followed by Bonferroni’s test vs. Scr-shRNA; n = 4. (D-E) Pard3 protein expression levels were analyzed by western blot, ***P < 0.001, ** P < 0.01, One-Way ANOVA followed by Bonferroni’s test vs. Scr-shRNA; n = 5. (F) Co-localization of Pard3 and the junctional protein ZO-1 was assessed by co-immunofluorescence staining. Pard3 was visualized with Alexa Fluor 647 (red), ZO-1 with Alexa Fluor 488 (green), and nuclei with DAPI(Blue). (G) Schematic of the pLV-Pard3-GFP lentiviral construct driven by the Ubc promoter. Construct integrity was confirmed by sequencing and western blot analysis. (H) Fibroblasts with shRNA-mediated knockdown of NDR1/2 were infected with Pard3- GFP–expressing lentivirus and enriched by FACS. Pard3 localization was visualized by GFP in both live-cell and fixed-cell imaging. The white dashed line indicates the scratch wound edge. Scale bars in (A, F, H): 20 µm.

Article Snippet: After blocking, the slides were incubated with primary antibodies overnight 4°C with antibodies including mouse anti-Vinculin (1: 300, Sigma, V9131), rabbit anti-GM130 (1:1000, CST, 12480T), rabbit anti- Pard3(1:400, Proteintech, 11085-1-AP), mouse anti-Zo-1 (1:500, Proteintech, 66452-1-Ig).

Techniques: Immunofluorescence, shRNA, Expressing, Western Blot, Staining, Construct, Sequencing, Knockdown, Infection, Imaging

Journal: bioRxiv

Article Title: NDR1/2 kinases regulate cell polarization and cell motility through Cdc42 GTPase and Pard3 signaling in mammalian cells

doi: 10.1101/2025.10.30.685405

Figure Lengend Snippet: (A) Schematic of Pard3 (top) highlighting the consensus NDR kinase phosphorylation motif (H.R..[S/T]); The N- terminal region of Pard3 (tagged with Myc at the N-terminus and 6×His at the C- terminus) contains the consensus phosphorylation site at Ser144. Wild-type (WT) and S144A mutant constructs were cloned into the pET22b backbone for inducible expression in E. coli DE3 cells (bottom). (B) Fibroblasts stably expressing lentivirus encoding GFP alone were subjected to wound-healing assays as controls; ****P < 0.0001; Two-Way ANOVA followed by Tukey’s post hoc tests, n = 40. (C-F) Validation of Pard3 phosphorylation at Ser144 by in vitro kinase assays. NDR1 and NDR2 (WT or kinase-dead [KD], , K118A for NDR1 and K119A for NDR2) were purified from HEK293T cells and incubated with purified WT N-Pard3 or S144A-mutant N-Pard3 protein. Reactions were performed in the presence of ATP-γ-S, and thiophosphorylation was detected by western blot using an anti–thiophosphate ester antibody, ***P < 0.001, **P < 0.01; One-Way ANOVA followed by Dunnett’s post hoc tests, n = 3. (G-H) Rescue wound-healing assays were performed to evaluate the effect of exogenous Pard3 or the Pard3-S144A mutant on fibroblast migration following NDR1/2 knockdown over 20 h, ****P < 0.0001, ***P < 0.001, *P < 0.05; Two-Way ANOVA followed by Tukey’s post hoc tests, n = 40-46.

Article Snippet: After blocking, the slides were incubated with primary antibodies overnight 4°C with antibodies including mouse anti-Vinculin (1: 300, Sigma, V9131), rabbit anti-GM130 (1:1000, CST, 12480T), rabbit anti- Pard3(1:400, Proteintech, 11085-1-AP), mouse anti-Zo-1 (1:500, Proteintech, 66452-1-Ig).

Techniques: Phospho-proteomics, Mutagenesis, Construct, Clone Assay, Expressing, Stable Transfection, Biomarker Discovery, In Vitro, Purification, Incubation, Western Blot, Migration, Knockdown

Journal: Oncogene

Article Title: EphA2 regulates vascular permeability and prostate cancer metastasis via modulation of cell junction protein phosphorylation

doi: 10.1038/s41388-024-03206-x

Figure Lengend Snippet: a , b Proteome Profiler Human Phospho-Kinase Array and western blot analysis validating pathway regulation downstream of EphA2 kinase activation in PC-3 cells. PC-3 cells were left untreated or incubated with clustered Fc control (Fc) or clustered ephrin-A1-Fc (A1) for 20 min to activate the EphA2 receptor. Representative images of array and western blot membranes are shown. β-actin is the western blot loading control. Increased PARD3 tyrosine phosphorylation was shown by immunoprecipitating PARD3 and subsequent probing with an anti-phospho-tyrosine antibody. Bar graph shows the quantification of the Proteome Profiler array as log2(fold change (efnA1-Fc:Fc control)). Mean ± SE, ( n = 4 biological replicates). c Western blot analysis assessing EphA2-signaling in a panel of prostate, breast, colon and brain cancer cell lines. Cell lines were incubated with clustered Fc control (Fc) or clustered ephrin-A1-Fc (A1) for 20 min. SHB phosphorylation on Y246 is consistently upregulated by EphA2 receptor activation across all tested cell lines. Downregulation of Afadin (S1718/S1799) and NDRG1 was observed only in cell lines which responded with a robust downregulation of Akt phosphorylation on S473 (PC-3, MDA-MB-231, BT-519, HCT-116 and MN1).

Article Snippet: Anti-PARD3 antibody (Thermo Fisher Scientific #11085-1-AP) was bound to Protein G Sepharose beads (1 h, 4 °C) and the unbound antibody was removed by washing with lysis buffer.

Techniques: Western Blot, Activation Assay, Incubation, Control