Journal: bioRxiv

Article Title: Drebrin forms a cortical hub that connects actin, microtubules, and clathrin for endocytic transport of β2 integrin

doi: 10.64898/2026.03.12.711275

Figure Lengend Snippet: (a) Time series of micrographs of primary human macrophages treated with control siRNA or drebrin siRNA, incubated with biotin to label surface proteins and with AlexaFluor488-streptavidin to visualize and quantify endocytosis of biotinylated proteins. Black outlines indicate cells with a high expression of drebrin, while red outlines mark cells with low expression. Note reduced streptavidin signal intensity (i.e. endocytosed surface proteins) in cells showing low drebrin expression and vice versa. (b) Quantification of streptavidin signal intensity, during endocytosis, in cells treated with control siRNA or drebrin siRNA. Signals are normalized to the mean intensity signal of the respective control. N= 49 cells from 4 independent donors, ****P<0.001, according to 2way ANOVA test. Bar: 10 µm. (c) Endocytosis of biotinylated surface proteins from cells treated with control siRNA or drebrin siRNA with representative Western blots showing β1, β2, β3 integrins, and α-tubulin as loading control. Time since start of the experiment is indicated in min. (d-f) Evaluation of signal intensities of blots from (c) Signals are normalized to mean intensity of drebrin signal of respective control. N= 2 independent donors, *P<0.05, according to multiple unpaired T-tests. (g) Analysis of total cellular expression levels of target proteins in primary human macrophages treated with drebrin siRNA for 72 hrs: N= at least 2 samples from independent donors; ns, according to One-way ANOVA test. (h) Western blot of anti-GFP immunoprecipitations from lysates of macrophages expressing drebrin-GFP or GFP empty vector as control, lysed with mild or stringent lysis buffers, and developed with anti-β1, β2, or β3 integrin antibodies. Note prominent coprecipitation of β2 integrin in both conditions and faint coprecipitation of β3 integrin only in mild condition. (i) Western blot analysis of cross-immunoprecipitations of endogenous dynamin or drebrin from macrophage lysates, with whole cell lysate (WCL) as control. Note absence of drebrin in anti-dynamin IP and vice versa. (j) Western blot analysis of whole cell lysates from HUVEC or primary human macrophages (left), and immunopreciptations from human macrophage lysates using control IgG or anti-drebrin antibody, probed for clathrin heavy chain, drebrin, and caveolin-1. Note detection of caveolin-1 in lysates from HUVEC, but not from macrophages. Also note co-precipitation of clathrin heavy chain with drebrin. Molecular weight in kDa is indicated on the right for (c,h-j).

Article Snippet: Following fixation, samples were washed three times with DPBS, regularly permeabilized for 5 min with 0.5 % Triton X-100, blocked for 1 hr with 2% BSA and 5% NGS, incubated for 90 min with blocking solution containing 1:400 AlexaFluor488-streptavidin (ThermoFisher Scientific, Cat# S32354), 1:400 AlexaFluor568-phalloidin (ThermoFisher Scientific, Cat# A12380), 1:1000 DAPI (ThermoFisher Scientific, Cat# 62248) and 1:200 anti-drebrin polyclonal Ab (ThermoFisher Scientific, Cat# PA5-84067), washed with 0.05% Triton X-100 (3x5 min) and then incubated for 60 min with blocking solution containing AlexaFluor647-anti-rabbit Ab (ThermoFisher Scientific, Cat# A-21246) before 3x5 min washes with 0.05% Triton X and mounting with Fluoromount-G medium (ThermoFisher Scientific, Cat# 00-4958-02).

Techniques: Control, Incubation, Expressing, Western Blot, Plasmid Preparation, Lysis, Molecular Weight