Journal: iScience

Article Title: FMRP-dependent translational control negatively regulates adapter protein complex 2-mediated endocytosis

doi: 10.1016/j.isci.2025.113062

Figure Lengend Snippet: FMRP negatively regulates the subcellular expression of AP-2 subunits (A) The subunits of three adapter protein complexes (AP1G1, AP2B1, and AP3D1), a postsynaptic marker (PSD95), a glial cell marker (GFAP), and GAPDH were examined in whole mouse brain lysates (INP) and synaptoneurosome (SNS) samples from mouse brain (postnatal day one). (B) Expression of PSD95, GFAP, and AP complexes in input (INP) and synaptoneurosomes (SNS). Signals were normalized to GAPDH. (C) AP-2 subunits, AP2A1 and AP2B1, in SNS samples prepared from eight pairs of WT and Fmr1 KO mouse brains are shown by western blot. (D) Expression levels of AP2A1 and AP2B1 in SNS samples from WT and Fmr1 KO mice. Data are represented as mean ± SEM. Unpaired t-test, N = 8, ∗ p < 0.05 and ∗∗ p < 0.01. (E) Representative images of cultured mouse cortical neurons (DIV12) from WT or Fmr1 KO mice. Immunofluorescence for AP2B1 overlaid with PSD95 and MAP2 to show the dendritic and synapse morphology. Enlarged boxes show dendritic segments of selected neurons. Scale bars, 40 μm. (F) Quantitative analysis of AP2B1 intensity and postsynaptic localization in soma and dendritic regions. Data are represented as mean ± SEM. Paired t-test, N = 3, ∗ p < 0.05 and ∗∗∗ p < 0.001.

Article Snippet: The following FAM probes: Ap2a1 (ThermoFisher Mm00475919_m1), or Ap2b1 (ThermoFisher Mm00551136_m1), were combined with VIC probe Gapdh (ThermoFisher 4326317E).

Techniques: Expressing, Marker, Western Blot, Cell Culture, Immunofluorescence

Journal: iScience

Article Title: FMRP-dependent translational control negatively regulates adapter protein complex 2-mediated endocytosis

doi: 10.1016/j.isci.2025.113062

Figure Lengend Snippet: FMRP associates with Ap2a1 and Ap2b1 mRNAs and represses nascent protein translation (A) mRNA levels of Ap2a1 and Ap2b1 in WT and Fmr1 KO mouse brains. Gapdh mRNA was used as internal control. Data are represented as mean ± SEM. Unpaired t-test, N = 8. (B) FMRP eCLIP binding sites within the mRNA sequences of Ap2a1 and Ap2b1 in E13.5 mouse brains. The intact mRNA sequences of Ap2a1 and Ap2b1 , including their 3′UTR regions, are presented, with peaks indicating the read densities of predicted binding sites within these regions. Size-matched input (SMI) represents the background reading. (C) RNA-immunoprecipitation (RIP) of FMRP-mRNA complex from mouse brain lysates. Confirmation of FMRP immunoprecipitation by western blot. (D) qPCR analysis of FMRP-mRNA complexes. Both Ap2a1 and Ap2b1 mRNAs were detected by qPCR. Psd95 and Map1b mRNAs were used as positive controls and Gapdh mRNA as a negative control. Data are represented as mean ± SEM. Paired t-test, N = 3, ∗∗∗∗ p < 0.0001. (E) Newly synthesized AP2B1 was detected by puro-PLA. WT or Fmr1 KO cortical neurons were treated with puromycin for 5 min before fixation, and the puro-PLA signal show nascent synthesis of AP2B1 within the 5 min time frame. Scale bars, 20 μm. (F) Nascent synthesis of AP2B1 in the soma (left) and dendritic (right) regions of WT and Fmr1 KO neurons. Data are represented as mean ± SEM. Paired t - test, N = 4, ∗ p < 0.05.

Article Snippet: The following FAM probes: Ap2a1 (ThermoFisher Mm00475919_m1), or Ap2b1 (ThermoFisher Mm00551136_m1), were combined with VIC probe Gapdh (ThermoFisher 4326317E).

Techniques: Control, Binding Assay, RNA Immunoprecipitation, Immunoprecipitation, Western Blot, Negative Control, Synthesized

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

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: Primary antibodies against AP2A1 (1:100, Novus Biologics, NB600-1545), miP-PSTPIP2 (5 μg/ml, Eurogentec, Seraing, Belgium) , control rabbit IgG (5 μg/ml, Millipore, NI01) or phalloidin-Alexa Fluor 633 (1:300, Invitrogen, A22284) were diluted in PBS containing 0.5% horse serum and 0.01% Triton X-100, and incubated for 2 hours at room temperature.

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: Primary antibodies against AP2A1 (1:100, Novus Biologics, NB600-1545), miP-PSTPIP2 (5 μg/ml, Eurogentec, Seraing, Belgium) , control rabbit IgG (5 μg/ml, Millipore, NI01) or phalloidin-Alexa Fluor 633 (1:300, Invitrogen, A22284) were diluted in PBS containing 0.5% horse serum and 0.01% Triton X-100, and incubated for 2 hours at room temperature.

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