anti-arc rabbit polyclonal affinity purified antibody #156 003  (Synaptic Systems)

Journal: eNeuro

Article Title: Activity-Regulated Cytoskeleton-Associated Protein Controls AMPAR Endocytosis through a Direct Interaction with Clathrin-Adaptor Protein 2 1 2 3

doi: 10.1523/ENEURO.0144-15.2016

Figure Lengend Snippet: Arc–AP-2 interaction regulates GluA1 endocytosis. A , B , Representative blots showing that Arc (WT), but not the Arc (W197A) mutant, facilitates GluA1, but not GluA2 endocytosis. H4 neuroglioma cells were transfected with plasmids encoding myc-GluA1 ( A ) or myc-GluA2 ( B ) in combination with either: empty pCIneo vector, pCIneo Arc (WT) , or pCIneo Arc (W197A) . Western blot band densitometry analysis showing that: ( A ) Arc (WT) , but not Arc (W197A) , promotes a significant reduction in surface expression of GluA1 subunits (control: 60.46 ± 2.97%; Arc (WT) : 38.55 ± 7.44%; Arc (W197A) : 50.18 ± 8.34%. Error bars represent mean ± SEM ( n = 3 independent experiments). * p <0.05 using one-way ANOVA followed by Tukeýs post-test. B , Arc (WT) does not promote any changes in surface expression of either GluA2 subunits (control: 132.9 ± 26.66%; Arc (WT ): 133.2 ± 21,78%; Arc (W197A) : 143.9 ± 38.43%) or EGF receptor (control: 51.35 ± 10.43%; Arc (WT) : 38.93 ± 8.66%; Arc (W197A) : 41.59 ± 8.8%). Error bars represent mean ± SEM ( n = 4 independent experiments).Ten percent of the protein lysate used for incubate the beads was loaded in the input lane. GAPDH was used as loading controls. C – F , C – G , H4 cells coexpressing myc-GluA1 with either mCherry construct alone ( C ), mCherry-Arc (WT) ( D ), or mCherry-Arc (W197A) ( E ). Surface myc-GluA1 (non-permeabilized cells, green channel) was identified using mouse anti-myc antibody followed by AlexaFluor 488 secondary antibody and internal myc-GluA1 (permeabilized, magenta channel) was identified by polyclonal rabbit anti-myc antibody followed by AlexaFluor 647 secondary antibody. F , G , The mean florescence intensity (MFI) of AlexaFluor 488 (surface my-GluA1) and mCherry (red channel) were calculated using confocal Z-projection images to quantify the pixel intensity of surface myc-GluA1 and mCherry (total protein expression). F , Ratio of averaged MFI between surface (488)/total protein (mCherry) for control cells ( n =59 cells) was set to 100% to facilitate comparison. Note that the ratio for surface GluA1 is significantly reduced in cells expressing mCherry-Arc (WT) (34.68 ± 3.13%; n = 60 cells) compared with cells expressing mCherry construct alone. Importantly, this reduction is absent in cells expressing the mCherry-Arc (W197A) construct (114.80 ± 13.08%; n = 42 cells. G , Bar chart plotting the averaged MFI expression levels of mCherry-Arc (WT) and mCherry-Arc (W197A) compared with mCherry expression. Values are mean ± SEM ( n =3 independent experiments). * p <0.05, ***p<0.005 using one-way ANOVA followed by Tukeýs post-test. Scale bar, 10 μm. H , Representative blot and bar chart plotting bands densitometry analysis of Arc expression protein in H4 cells transfected with equal amounts of mCherry-Arc (WT) , mCherry-Arc (W197A) , or mCherry-Arc (195-199A) plasmids. Note the similar levels Arc protein expression between samples. Values are mean ± SEM ( n =3 independent experiments).

Article Snippet: Five-hundred micrograms of protein, making 500 μl of final volume, was incubated with 1 μg of rabbit polyclonal anti-Arc antibody (Synaptic Systems, 156-003) and 15 μl of prewashed protein G agarose beads (Upstate-Millipore, 16-266) and rotated for 3 h at 4°C.

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Western Blot, Expressing, Control, Construct, Comparison