Journal: Oncogenesis

Article Title: Regulation of cargo transfer between ESCRT-0 and ESCRT-I complexes by flotillin-1 during endosomal sorting of ubiquitinated cargo

doi: 10.1038/oncsis.2017.47

Figure Lengend Snippet: EGFR degradation is impaired in the absence of flotillin-1.( a , b ) Control siRNA, flotillin-1 (F1-siRNAa and -b) and ( c , d ) control HeLa or flotillin-1-knockout cells (transfected with flotillin-1-EGFP or not) were starved overnight and stimulated with 100 ng/ml EGF for 60 min, fixed with methanol and immunostained for EGFR. ( b ) The amount of undegraded EGFR in flotillin-1-knockdown cells was quantified as fluorescence intensity in arbitrary units using the ImageJ Software. A significant increase of EGFR signal was observed in flotillin-knockdown cells as compared with the control. ( d ) EGFR amount after 60 min was quantified as in c , and the data show a rescue of EGFR degradation upon flotillin-1-EGFP expression (cells marked with *, images for GFP in ). The data in c , d are shown as mean±s.d. Cells from three independent experiments were evaluated (control siRNA cells: n =136, F1-siRNAa: n =135 and F1-siRNAb: n =149; control HeLa: n =358, flotillin-1 KO: n =181, rescue cells: n =252). Statistical analysis was performed with one-way analysis of variance (ANOVA) and Bonferroni post-test in comparison with the control. *** P <0.001. Scale bar in ( a , c , e ): 10 μm. ( e ) Flotillin-1-knockdown cells (F1-siRNAb) were treated as in a and immunostained for EGFR (green) and Rab5, Hrs or LAMP1 (red).

Article Snippet: Mouse monoclonal antibody against Hrs (sc-271455, clone C-7; WB: 1:1000), CD63/LAMP3 (sc-5275; IF: 1:200), ubiquitin (sc-8017, clone P4D1; WB: 1:1000, IF: 1:100) and EGFR (sc-120, clone 528; IF: 1:100, WB: 1:1000) as well as rabbit polyclonal antibodies against Hrs (sc-30221, clone M-79; IF: 1:40, WB: 1:1000; IP: 1:100), Rab5 (sc-46692; IF: 1:200) and c-myc (sc-789; IP: 1:300) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Control, Knock-Out, Transfection, Knockdown, Fluorescence, Software, Expressing, Comparison

Journal: Oncogenesis

Article Title: Regulation of cargo transfer between ESCRT-0 and ESCRT-I complexes by flotillin-1 during endosomal sorting of ubiquitinated cargo

doi: 10.1038/oncsis.2017.47

Figure Lengend Snippet: Endosomal morphology is altered in flotillin-1-knockdown/knockout cells, but endosomal domains persist.( a ) Control and flotillin-1-knockout cells were chased for 2 h with Fe-EGF, fixed and analysed by transmission electron microscopy. Note the enlarged endosomes in flotillin-1-knockdown cells. Scale bar: 500 nm. ( b ) Control siRNA and flotillin-1-knockdown cells were stimulated with EGF for 30 min and labelled with an anti-mouse LAMP3 antibody detected with a secondary Alexa Fluor 647-coupled antibody. The specimens were analysed with Leica SR GSD 3D microscope. Scale bar: 2 μm. ( c ) Rab5-Q79L-GFP-expressing HeLa cells were immunostained for endogenous flotillin-2 and Hrs, which show a limited colocalization in the enlarged endosomes. Scale bar: 10 μm. ( d ) Rab5-Q79L-GFP-expressing HeLa and flotillin-1-knockout cells were immunostained for endogenous CHC and Hrs, which are localised in endosomal domains in both cell types and show some colocalization. Scale bar: 10 μm.

Article Snippet: Mouse monoclonal antibody against Hrs (sc-271455, clone C-7; WB: 1:1000), CD63/LAMP3 (sc-5275; IF: 1:200), ubiquitin (sc-8017, clone P4D1; WB: 1:1000, IF: 1:100) and EGFR (sc-120, clone 528; IF: 1:100, WB: 1:1000) as well as rabbit polyclonal antibodies against Hrs (sc-30221, clone M-79; IF: 1:40, WB: 1:1000; IP: 1:100), Rab5 (sc-46692; IF: 1:200) and c-myc (sc-789; IP: 1:300) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Knockdown, Knock-Out, Control, Transmission Assay, Electron Microscopy, Microscopy, Expressing

Journal: Nature Communications

Article Title: IFITM proteins promote SARS-CoV-2 infection and are targets for virus inhibition in vitro

doi: 10.1038/s41467-021-24817-y

Figure Lengend Snippet: a Quantification and exemplary images of a PLA between SARS-CoV-2 Spike and ACE2 in Calu-3 depleted of IFITM1, IFITM2, or IFITM3 and infected with genuine SARS-CoV-2. Bars represent the mean of four independent experiments (100 cells ±SEM), two-sided Wilcoxon matched-pairs test, **** p < 0.0001. b Quantification and exemplary images of a PLA between Spike and ACE2 in Calu-3 cells depleted of IFITM2 and infected with SARS-CoV-2 virus on ice for 2 h and then incubated for 15 min at 37 °C. Bars represent the mean of four independent experiments (300 cells, ±SEM), two-sided Wilcoxon matched-pairs test, **** p < 0.0001. c Quantification and exemplary images of a PLA assay between Spike and RAB5A as in ( c ). Bars represent the mean of four independent experiments (400 cells, ±SEM), two-sided Wilcoxon matched-pairs test, **** p < 0.0001. DAPI (blue), nuclei. PLA signal (yellow). Scale bar, 20 µm. d Summary of the quantification shown in panels ( b ) (Spike-ACE2) and ( c ) (Spike-RAB5α) proximity upon SARS-CoV-2 infection. Individual data points and statistics are provided in panels ( b ) and ( c ). e Exemplary images of the colocalization (white) of PLA foci (red) indicating SARS-CoV-2 Spike and IFITM2 with early (EEA1, green, upper panel) or late endosomal markers (Rab7a, green, lower panel) in Calu-3 cells. Cells were infected with SARS-CoV-2 for 2 h at 4 °C or 2 h at 4 °C and 15 min at 37 °C as indicated. DAPI (blue), nuclei. f Quantification of the percentage of colocalization between the PLA signal and the indicated endosomal markers in 225 × 225 µm images. Bars represent the mean of four individual images (±SEM), unpaired t test, ** p = 0.0031. g Quantification of the combined PLA (IFITM2/Spike) in ( e ). Bars represent the mean of 120 cells (±SEM) over two independent experiments. The experiment was replicated twice to similar results. Two-sided Wilcoxon matched-pairs test, **** p < 0.0001. a – c , e Scale bars, 20 μm.

Article Snippet: For staining following antibodies were used: IFITM1 (α-IFITM1 Cell Signaling 13126S), IFITM2 (α-IFITM2 Abcam 236735), IFITM3 (α-IFITM3 Cell Signaling 59212S), SARS Spike CoV-2 (SARS-CoV / SARS-CoV-2 (COVID-19) spike antibody [1A9], GTX-GTX632604), Rab5 alpha (Rab5 (RAB5A) Goat Polyclonal Antibody Origene AB0009-200) and ACE2 (Rabbit polyclonal anti-ACE2 Abcam, ab166755).

Techniques: Infection, Incubation

Journal: Journal of pharmaceutical analysis

Article Title: Monoclonal antibody targeting mu-opioid receptor attenuates morphine tolerance via enhancing morphine-induced receptor endocytosis.

doi: 10.1016/j.jpha.2023.06.008

Figure Lengend Snippet: Fig. 1. Effects of 3A5C7 monoclonal antibody (mAb) on morphine-induced mu-opioid receptor (MOR) endocytosis from cell membrane to cytoplasm. (A) Immunofluorescence staining indicated the endocytosis of MOR in HEK293T-MOR cells treated with morphine and 3A5C7 mAb. (B) Immunofluorescence staining indicated the endocytosis of MOR in SH- SY5Y cells treated with morphine and 3A5C7 mAb. (C) Immunofluorescence staining manifested the colocalization of MOR and Rab5 in HEK293T-MOR cells subjected to 3A5C7 mAb and morphine. (D, E) Flow cytometry showed the endocytosis of MOR in HEK293T-MOR cells co-treated with morphine and 3A5C7 mAb. (F, G) Flow cytometry showed the

Article Snippet: Anti-MOR antibody (ab10275; Abcam) and anti-Rab5 antibody (orb153348; Biorbyt) were used as primary antibodies, and Alexa 488-conjugated donkey anti-rabbit IgG and Alexa 555-conjugated donkey anti-goat IgG (Thermo Fisher endocytosis of MOR in SH-SY5Y cells co-treated with morphine and 3A5C7 mAb. (H) Immuno and 3A5C7 mAb.

Techniques: Membrane, Staining, Flow Cytometry

Journal: Journal of pharmaceutical analysis

Article Title: Monoclonal antibody targeting mu-opioid receptor attenuates morphine tolerance via enhancing morphine-induced receptor endocytosis.

doi: 10.1016/j.jpha.2023.06.008

Figure Lengend Snippet: Fig. 6. 3A5C7 monoclonal antibody (mAb) attenuates morphine antinociceptive tolerance and physical dependence in mice. (A) Western blots indicating the cross specificity of 3A5C7 mAb against mu-opioid receptor (MOR) in the cortex, cerebellum, and hippocampus from C57/B6 mice. (B) Experiment flowchart for testing the effects of chronically administered 3A5C7 mAb on morphine tolerance and dependence. (C) The effects of chronically administered 3A5C7 mAb on the antinociceptive effects of morphine in mice measured by hotplate test. (D) The body weight changes of morphine-tolerant mice. Two-way analysis of variance with Bonferroni's post hoc tests were used for statistical analysis (n ¼ 6e7 mice in each group). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, morphine þ mAb group vs. morphine group. #P < 0.05, ##P < 0.01, and ###P < 0.001, morphine þ mAb group vs. morphine þ normal IgG (NIg) group. (E) Representative immunofluorescence staining of MOR and Rab5 in the mouse dorsal root ganglions (DRGs) after

Article Snippet: Anti-MOR antibody (ab10275; Abcam) and anti-Rab5 antibody (orb153348; Biorbyt) were used as primary antibodies, and Alexa 488-conjugated donkey anti-rabbit IgG and Alexa 555-conjugated donkey anti-goat IgG (Thermo Fisher endocytosis of MOR in SH-SY5Y cells co-treated with morphine and 3A5C7 mAb. (H) Immuno and 3A5C7 mAb.

Techniques: Western Blot, Staining

Journal: Journal of pharmaceutical analysis

Article Title: Monoclonal antibody targeting mu-opioid receptor attenuates morphine tolerance via enhancing morphine-induced receptor endocytosis.

doi: 10.1016/j.jpha.2023.06.008

Figure Lengend Snippet: Fig. 7. 3A5C7 monoclonal antibody (mAb) attenuates morphine antinociceptive tolerance via G protein-coupled receptor kinase 2 (GRK2)/b-arrestin2 pathway in mice. (A) Immunoblot showing that GRK2 was knocked-down by short hairpin ribonucleic acid (shRNA) in dorsal root ganglions (DRGs). (B) Quantification of the protein level of GRK2 in Fig. 7A. (C) Immunoblot showing that b-arrestin2 was knocked-down by shRNA in DRGs. (D) Quantification of the protein level of b-arrestin2 in Fig. 7C. (E) Hotplate tests demonstrating the effects of GRK2 or b-arrestin2 knockdown on the anti-tolerance efficacy of mAb 3A5C7. Two-way analysis of variance (ANOVA) with Bonferroni's post hoc tests were used for statistical analysis. **P < 0.01 and ****P < 0.0001, sh-NC þ morphine þ mAb group vs. sh-NC þ morphine group; ###P < 0.001 and ####P < 0.0001, sh- NC þ morphine þ mAb group vs. sh-GRK2 þ morphine þ mAb group; &P < 0.05 and &&&P < 0.001, sh-NC þ morphine þ mAb group vs. sh-b-arrestin2 þ morphine þ mAb group (n ¼ 5 mice in each group). (F) Representative immunofluorescence images of MOR and Rab5 for each treatment in mouse DRGs (n ¼ 3 mice in each group). (G) The protein levels of hippocampal protein kinase A (PKA) from mice in each treatment group. (H, I) Quantification of the relative protein levels of PKA from Fig. 7G. One-way ANOVA with Bonferroni's post hoc tests were used for statistical analysis. **P < 0.01 and ***P < 0.001. (J) Inhibitory effects of acute 3A5C7 mAb administration on naloxone-precipitated withdrawal jumping were reduced in GRK2- and b-arrestin2-knockdown mice. One-way ANOVA with Bonferroni's post hoc tests were used for statistical analysis. ****P < 0.0001 vs. sh-NC þ morphine group; ####P < 0.0001 vs. sh-NC þ morphine þ mAb group (n ¼ 5 mice in each group). Data were presented as the mean ± standard error of mean. sh-NC: control shRNA; sh-GRK2: shRNA for GRK2; sh-b-arrestin2: shRNA for b-arrestin2; MPE: maximum possible effect; GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Anti-MOR antibody (ab10275; Abcam) and anti-Rab5 antibody (orb153348; Biorbyt) were used as primary antibodies, and Alexa 488-conjugated donkey anti-rabbit IgG and Alexa 555-conjugated donkey anti-goat IgG (Thermo Fisher endocytosis of MOR in SH-SY5Y cells co-treated with morphine and 3A5C7 mAb. (H) Immuno and 3A5C7 mAb.

Techniques: Western Blot, shRNA, Knockdown, Control

Journal: Bioactive Materials

Article Title: Matrix stiffness exacerbates the proinflammatory responses of vascular smooth muscle cell through the DDR1-DNMT1 mechanotransduction axis

doi: 10.1016/j.bioactmat.2022.01.012

Figure Lengend Snippet: Substrate stiffness-induced DDR1 activation is independent of collagen binding. (A) to (C) Western blot assay to determine phosphorylation of DDR1 at tyrosine 792 (p-DDR1) and the total DDR1 expression. GAPDH served as an internal control. Shown are representative blots from at least 3 independent experiments. Semi-quantification (in the lower panels) of indicated protein was performed by using the ImageJ software based on the analysis of the gray band intensity. (A) The p-DDR1 and total DDR1 levels in SMCs cultured for 24 h on collagen type I (Col) coated-polyacrylamide (PA) gels with a stiffness of 2 (soft) or 20 (stiff) kPa. (B) The p-DDR1 levels in SMCs grown on Col-coated gels for 24 h in the presence of the DDR1: Fc peptides. (C) The p-DDR1 and total DDR1 levels in SMCs cultured for 24 h on fibronectin (Fn)-coated PA gels. (D) Representative stimulated emission depletion microscopy (STED) images of DDR1 and Rab5A immunofluorescence in SMCs on Fn-coated gels. Cells were either pretreated with DDR1-IN-1 or DMSO. (E) Schematic diagram illustrating measuring the interaction between the collagen and the surfaces of SMCs by atomic force microscopy (AFM). (F) AFM to determine the adhesion probability and rupture forces between the Col- or BSA-coated probe and the cell surface. Each dot represents one cell. (G) and (H) AFM to determine the adhesion probability and rupture forces between the DDR1 ligand (Col) and the cell surface. In (G) , each dot represents an independent experiment, and in (H) , each dot represents the rupture force of a single bond between Col and the cell surface. (I) AFM to determine the adhesion probability in cells that were pretreated with DDR1-IN-1 or DMSO and cultured on Fn-coated gels. Each dot represents an independent experiment. (J) Western blot assay to determine DDR1 and GAPDH in cell lysates prepared using a non-reducing condition. (K) Quantitative RT-PCR to determine the expressions of DDR1 targeted genes in SMCs on Fn-coated gels. Each dot represents an independent experiment. * P < 0.05 vs. the indicated group.

Article Snippet: Rabbit polyclonal antibodies against DNMT1, p53, and DDR1, and mouse monoclonal antibody against Rab5A were purchased from Proteintech.

Techniques: Activation Assay, Binding Assay, Western Blot, Phospho-proteomics, Expressing, Control, Software, Cell Culture, Microscopy, Immunofluorescence, Quantitative RT-PCR

Journal: eLife

Article Title: Critical role for Piccolo in synaptic vesicle retrieval

doi: 10.7554/eLife.46629

Figure Lengend Snippet:

Article Snippet: Antibody , anti-Rab5 (mouse monoclonal) , Synaptic systems , RRID: AB_887773 , (1:500).

Techniques: Recombinant, Subcloning, Plasmid Preparation, Sequencing