Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Hsc70 phosphorylation patterns and calmodulin regulate AP2 Clathrin-Coated-Vesicle life span for cell adhesion protein transport.

doi: 10.1016/j.bbamcr.2023.119611

Figure Lengend Snippet: Fig. 1. Summary of the AP1/σ1B ko induced changes in synaptic AP2 CCV. A The two classes of synaptic AP2 CCV. Fast uncoating canCCV and slow uncoating wt stable CCV. Pool sizes of both are doubled in AP1/σ1B ko synapses. In addition, stable CCV are stabilized, named stCCV. B Summary of Hsc70 and co-chaperone protein levels relative to their wt levels in canCCV and stCCV (100 %) [15].

Article Snippet: Isolation of stable CCV from wt and of stCCV from ko mice: sucrose density gradient fractions containing the purified synaptic CCV were pooled, and incubated with protein G Sepharose bead slurry (Protein G Sepharose 4 Fast Flow GE Healthcare) at 4 ◦C for 1 h. Beads were pelleted at 800 x g and the supernatant was incubated with 5 μg of anti-Hsc70 mouse monoclonal antibody (Synaptic Systems, Göttingen, Ger) over night at 4 ◦C.

Techniques:

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Hsc70 phosphorylation patterns and calmodulin regulate AP2 Clathrin-Coated-Vesicle life span for cell adhesion protein transport.

doi: 10.1016/j.bbamcr.2023.119611

Figure Lengend Snippet: Fig. 3. A. Changes in the amounts of the CCV Hsc70 NEF Hsp110 and of the Hsc70 co-chaperone Hsp90 in AP1/σ1B ko brains. Numbers below each box indicate the numbers of independent biological samples. Representative semi-quantitative western-blot bands are shown below each box-plot diagram (see suppl. p.2–6). B. Summary of the changes in of Hsc70 and Hsp110 in canCCV and stCCV levels expressed in % per CCV and the Hsc70:Hsp110 ratios in canCCV and stCCV.

Article Snippet: Isolation of stable CCV from wt and of stCCV from ko mice: sucrose density gradient fractions containing the purified synaptic CCV were pooled, and incubated with protein G Sepharose bead slurry (Protein G Sepharose 4 Fast Flow GE Healthcare) at 4 ◦C for 1 h. Beads were pelleted at 800 x g and the supernatant was incubated with 5 μg of anti-Hsc70 mouse monoclonal antibody (Synaptic Systems, Göttingen, Ger) over night at 4 ◦C.

Techniques: Western Blot

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Hsc70 phosphorylation patterns and calmodulin regulate AP2 Clathrin-Coated-Vesicle life span for cell adhesion protein transport.

doi: 10.1016/j.bbamcr.2023.119611

Figure Lengend Snippet: Fig. 7. Summary of the changes in Hsc70 phosphorylation levels associated with the formation of AP2 canCCV with short, regular life spans and of AP2 stCCV with a stabilized vesicle protein coat and thus an increased life time.

Article Snippet: Isolation of stable CCV from wt and of stCCV from ko mice: sucrose density gradient fractions containing the purified synaptic CCV were pooled, and incubated with protein G Sepharose bead slurry (Protein G Sepharose 4 Fast Flow GE Healthcare) at 4 ◦C for 1 h. Beads were pelleted at 800 x g and the supernatant was incubated with 5 μg of anti-Hsc70 mouse monoclonal antibody (Synaptic Systems, Göttingen, Ger) over night at 4 ◦C.

Techniques: Phospho-proteomics

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Nkx2‐5 Is Expressed in Atherosclerotic Plaques and Attenuates Development of Atherosclerosis in Apolipoprotein E–Deficient Mice

doi: 10.1161/JAHA.116.004440

Figure Lengend Snippet: NK 2 homeobox 5 (Nkx2‐5) inhibits monocyte‐endothelial adhesion and decreases expression of adhesion molecules in early atherosclerosis. A, Upper panel: representative images used for quantification of peripheral blood monocytes ( PBMC ; green, calcein AM ) attached to aortic endothelial cells ( HAEC s, blue, DAPI ). Middle panel: effects of Nkx2‐5 on expression of vascular cell adhesion molecule‐1 ( VCAM ‐1) in endothelial cells (representative immunofluorescence image: green for VCAM ‐1 and blue for cell nuclei stained with DAPI ). Lower panel: effects of Nkx2‐5 on expression of E‐selectin in endothelial cells (representative immunofluorescence image: red for E‐selectin and blue for cell nuclei stained with DAPI ). B, Quantification of adhesion. The number of PBMC adhered to per 100 HAEC s was calculated. Results are expressed as a percent of values determined in the Ad‐ EV ‐treated group. Data represent the mean± SEM of 3 independent experiments. C, Representative immunoblot for intercellular adhesion molecule‐1 ( ICAM ‐1), VCAM ‐1, E‐selectin, and P‐selectin in endothelial cells infected with Ad‐ EV or Ad‐Nkx2‐5. D, Cross‐sections of early atherosclerotic lesions in aortic sinus were immunostained with antibodies against von Willebrand factor ( vWF ), VCAM ‐1, and E‐selectin. Staining with rabbit IgG isotype was used as the negative control. E, Quantification of histochemical staining of VCAM ‐1 and E‐selectin. Positive stained areas were quantified as a percentage of total plaque area or plaque endothelium. Data are expressed as mean± SEM (n=12 per group). AM indicates acetomethoxy; DAPI, 4′,6‐diamidino‐2‐phenylindole; HAECs, human aortic endothelial cells; IgG, immunoglobulin G; TNFα, tumor necrosis factor alpha.

Article Snippet: Fresh adult human PBMCs (PCS‐800‐011; ATCC) were labeled with calcein acetomethoxy (AM) dye: PBMCs were pelleted at 240 g for 10 minutes, resuspended in 1 mL of culture medium with 2.5 μmol/L of calcein AM from the kit, and incubated at 37°C (5% CO 2 ) for 30 minutes.

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Infection, Negative Control