Journal: Science Advances

Article Title: Bi-handed assembly chaperones regulate protein complex assembly through an intramolecular handover mechanism

doi: 10.1126/sciadv.adw9158

Figure Lengend Snippet: ( A ) Diagram of pull-down assays detecting AAGAB binding to α and σ2. His 6 -SUMO–tagged WT AAGAB was coexpressed with GST-tagged α (trunk domain, amino acids 1 to 621), untagged WT σ2, or both in E. coli . Lysates from E. coli expressing the indicated proteins were used for pull-down assays. ( B ) Representative Coomassie blue–stained gels (top) and immunoblots (bottom) showing the binding of His 6 -SUMO–tagged WT AAGAB to GST-tagged α, untagged σ2, or both, as depicted in (A). ( C ) Representative immunoblots showing the interaction of 3xFLAG-tagged AAGAB with HA-tagged α and σ2 in AAGAB KO HeLa cells. AAGAB was immunoprecipitated using anti-FLAG antibodies, and proteins in the immunoprecipitates were detected via immunoblotting. ( D ) AlphaFold-predicted structural models of the AAGAB-CTD:α dimer and the AAGAB-CTD:α:σ2 trimer. The dimer prediction was performed using the CTD (amino acids 258 to 315) of AAGAB and the α trunk domain, whereas the trimer prediction was performed using the CTD of AAGAB, the α trunk domain, and WT σ2. The PDB/CIF files of these structural models are included in datasets S1 and S3. ( E ) Diagram of a GST pull-down assay detecting the binding of AAGAB (WT, GD, or CTD) to α and σ2. ( F ) Representative Coomassie blue–stained gels showing the binding of AAGAB to GST-tagged α and untagged σ2 as depicted in (E). ( G and H ) Quantification of GST-tagged α and untagged σ2 based on Coomassie blue–stained gels from four independent experiments, including data shown in (F). Data are presented as mean ± SD. *** P < 0.001; n.s., P > 0.05 (calculated using one-way ANOVA with Holm-Sidak corrections). ( I ) Representative Coomassie blue–stained gels showing His 6 -SUMO–tagged AAGAB isolated using nickel beads from the samples described in (E). ( J ) Quantification of His 6 -SUMO–tagged AAGAB based on Coomassie blue–stained gels.

Article Snippet: The endogenous AP2 α subunit was labeled using anti–α-adaptin antibodies (Santa Cruz Biotechnology, #sc-17771, RRID: AB_2274034) and Alexa Fluor 568–conjugated secondary antibodies (Thermo Fisher Scientific, #A11004, RRID: AB_2534072) or Alexa Fluor 488–conjugated secondary antibodies (Thermo Fisher Scientific, #A11008, RRID: AB_142165).

Techniques: Binding Assay, Expressing, Staining, Western Blot, Immunoprecipitation, Pull Down Assay, Isolation

Journal: bioRxiv

Article Title: Microprotein miP-PSTPIP2 affects cytoskeleton dynamics to modulate endothelial cell endocytosis, barrier function and migration

doi: 10.1101/2025.11.07.687176

Figure Lengend Snippet: A-B, Volcano plot (A) and GO term enrichment analysis (STRING) (B) of the proteins physically associated with miP-PSTPIP2 (versus rabbit IgGs; CTL) in human endothelial cells treated with IL-1β; n=3 independent cell batches. The dashed lines mark the significance threshold (P value: 0.05). C, Representative confocal images showing miP-PSTPIP2 and F-actin in human endothelial cells. Nuclei were stained with DAPI. Scale bar: 10 µm D, Co-immunoprecipitation (IP) of miP-PSTPIP2 and β-actin or DAAM1 from human endothelial cell lysates. Similar results were obtained using 2 additional cell batches. E-F, Representative confocal images of the interaction (proximity ligation assay: PLA) between miP-PSTPIP2 and caveoilin-1 (CAV1) (E) or Clathrin Heavy Chain (CHC) (F) in human endothelial cells; Scale bar: 10 µm. Similar results were obtained in 3 additional cell batches. G, Co-immunoprecipitation (IP) of miP-PSTPIP2 and CAV1 or CHC from human endothelial cell lysates. Similar results were obtained using 3 additional cell batches. H, Representative confocal images of AP2A1 and miP-PSTPIP2 in human endothelial cells; Scale bar: 50 µm. Similar results were obtained in 3 additional cell batches.

Article Snippet: Membranes were incubated overnight with the following primary antibodies: AP2A1 (1:1000, Novus Biologics, Centennial, United States, NB600-1545), AP2M1 (1:1000, Cell Signalling, Danver, United States, #68196), Arp2 (1:2000, Proteintech, Rosemont, United States, 10922-1-AP), Arp3 (1:1000, Abcam, Cambridge, United Kingdom, ab49671), β-actin (1:1000, Sigma-Aldrich, Burlington, United States, A1978), CAV1 (1:1000, Cell Signaling, #3238), CHC (1:2000, BD Transduction, Franklin Lakes, United States, 610499), Cortactin (1:10000, Abcam, ab81208), DAAM1 (1:2000, Proteintech, 67287-1-Ig), GAPDH (1:300, Millipore, Burlington, United States, MAB374), LDLR (1:2000, Invitrogen, Waltham, United States, PA5-46985), Phospho-AP2M1 (1:1000, Cell Signaling, #7399), RhoA (1:1000, Santa Cruz, Dallas, United States, sc-179) and VASP (1:1000, BD Transduction, V40020).

Techniques: Staining, Immunoprecipitation, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Microprotein miP-PSTPIP2 affects cytoskeleton dynamics to modulate endothelial cell endocytosis, barrier function and migration

doi: 10.1101/2025.11.07.687176

Figure Lengend Snippet: A, Western blot analysis and quantification of clathrin heavy chain (CHC), low-density lipoprotein receptor (LDLR), Caveolin-1 (CAV1), AP-2 complex subunit alpha-1 (AP2A1), phospho (p-AP2M1) and total AP-2 complex subunit mu (AP2M1) expression in human endothelial cells overexpressing miP-PSTPIP2 (miP) or EGFP as control (CTL). β-Actin or GAPDH were used as loading controls; n=4 independent cell batches. C, Flow cytometry analysis of surface LDLR expression in human endothelial cells overexpressing miP-PSTPIP2 (miP) or EGFP (CTL). Data are shown as mean fluorescence intensity (MFI); n=4 independent cell batches. D, Representative Total Internal Reflection Fluorescence (TIRF) microscopy images of miP-PSTPIP2 and transferrin in human endothelial cells starved of serum overnight and subsequently incubated with AF555–transferrin for 1, 5 or 15 minutes. n=3 independent experiments; scale bar: 20 µm. All statistical analyses were performed using unpaired Student’s t-tests.

Article Snippet: Membranes were incubated overnight with the following primary antibodies: AP2A1 (1:1000, Novus Biologics, Centennial, United States, NB600-1545), AP2M1 (1:1000, Cell Signalling, Danver, United States, #68196), Arp2 (1:2000, Proteintech, Rosemont, United States, 10922-1-AP), Arp3 (1:1000, Abcam, Cambridge, United Kingdom, ab49671), β-actin (1:1000, Sigma-Aldrich, Burlington, United States, A1978), CAV1 (1:1000, Cell Signaling, #3238), CHC (1:2000, BD Transduction, Franklin Lakes, United States, 610499), Cortactin (1:10000, Abcam, ab81208), DAAM1 (1:2000, Proteintech, 67287-1-Ig), GAPDH (1:300, Millipore, Burlington, United States, MAB374), LDLR (1:2000, Invitrogen, Waltham, United States, PA5-46985), Phospho-AP2M1 (1:1000, Cell Signaling, #7399), RhoA (1:1000, Santa Cruz, Dallas, United States, sc-179) and VASP (1:1000, BD Transduction, V40020).

Techniques: Western Blot, Expressing, Control, Flow Cytometry, Fluorescence, Microscopy, Incubation