Journal: eLife

Article Title: The membrane-associated proteins FCHo and SGIP are allosteric activators of the AP2 clathrin adaptor complex

doi: 10.7554/eLife.03648

Figure Lengend Snippet: In ( A ) and ( B ) APA domains from FCHo homologs were expressed as HaloTag fusions in HEK293T cells ( Ce , C. elegans ; Mm , M. musculus ). ( A ) Silver-stained gel of affinity-purified proteins following proteolytic cleavage from the HaloTag. Arrows indicate bands of presumed identity. ( B ) The top ten human proteins purified using two different APA baits, as detected by MudPIT mass spectrometry. Nonspecific proteins also found in the control were removed. Values represent the mean % distributed Normalized Spectral Abundance Factor (dNSAF × 100) from three independent experiments. The values of all four AP2 subunits were totaled to determine the amount of complex in each sample. Multiple isoforms of alpha, beta, and phosphatidylinositol 5-phosphate 4-kinase type-2 (PIP4K2) were summed. WD repeat-containing protein 48 (WDR48), epidermal growth factor receptor substrate 15 (EPS15), adaptin ear-binding coat-associated protein 2 (NECAP2), and serine/threonine-protein kinase PLK1 (PLK1). See for complete results. ( C ) APA pulldowns using bacterially expressed proteins. Purified HaloTag with (HT + APA) and without (HT) the APA domain from mouse SGIP1 were incubated with purified AP2 fragments followed by TEV protease cleavage to release the APA bait. Silver-stained gel of the eluted proteins. Note that the alpha/sigma and beta/mu hemicomplexes are soluble in our hands and that the AP2 appendage (ear) domains exhibit non-specific binding in this assay. DOI: http://dx.doi.org/10.7554/eLife.03648.017

Article Snippet: For western blot analysis of FCHo1 interactors, 20 μl of purified complexes were electrophoresed, transferred, and blotted as described above using the following primary antibodies: mouse anti-alpha adaptin (610501; 1:2000; BD Biosciences, San Jose, CA), rabbit anti-AP2B1 (151961; 1:1000; Abcam), rabbit anti AP2M1 (75995; 1:1000; Abcam) and rabbit anti-AP2S1 (128950; 1:10000; Abcam).

Techniques: Staining, Affinity Purification, Purification, Mass Spectrometry, Binding Assay, Incubation

Journal: Scientific Reports

Article Title: Endocytosis and lysosomal degradation of GluA2/3 AMPARs in response to oxygen/glucose deprivation in hippocampal but not cortical neurons

doi: 10.1038/s41598-017-12534-w

Figure Lengend Snippet: GluA2 endocytosis and PICK1-AP2 interaction are increased by OGD/reperfusion in hippocampal, but not cortical neurons. ( a ) GluA2 is not endocytosed in cortical neurons following OGD/reperfusion. Neurons were live-labeled with anti-GluA2 antibodies prior to 20 min OGD and 15 min reperfusion. The intensities of the remaining surface (green) and internalized (red) endogenous GluA2 were measured after fixation and used to calculate the internalization index (internalized/surface + internalized). Representative confocal images of dendrites from cortical cultures are shown. Two-tailed Student’s t-test. ( b ) Endogenous GluA2 is not degraded following OGD and reperfusion in cultured cortical neurons. Cell lysates were analyzed by Western blotting. Graph shows total GluA2 levels following 20 min OGD and 0, 30 min, 1 h and 3 h reperfusion. One-way ANOVA followed by Dunnett’s test. Blots shown are cropped; full-length blots are shown in Supplementary Fig. . ( c ) OGD causes an increase in endogenous PICK1-AP2 interactions in hippocampal but not cortical neurons. Extracts of cultured hippocampal and cortical neurons were immunoprecipitated with PICK1 antibodies or control IgG. Proteins were detected with anti-PICK1 and anti-α-adaptin (AP2) antibodies. Input is 5% of offered protein. Two-tailed Student’s t-test. Blots shown are cropped; full-length blots are shown in Supplementary Fig. .

Article Snippet: The antibodies used were as follows: α-adaptin (Becton Dickinson); GluA1 (Millipore); GluA2 (rabbit Synaptic Systems, mouse Becton Dickinson); GluA3 (Alomone); LAMP1 (Abcam); PICK1 (rabbit Abcam, mouse Neuromab); tubulin (Sigma); Rab5 (Cell Signaling); Rab11 (Cell Signaling).

Techniques: Labeling, Two Tailed Test, Cell Culture, Western Blot, Immunoprecipitation, Control