Journal: Frontiers in Synaptic Neuroscience
Article Title: Cyfip1 Regulates SynGAP1 at Hippocampal Synapses
doi: 10.3389/fnsyn.2020.581714
Figure Lengend Snippet: Cyfip1 haploinsufficiency increases Rac1 activity. (A) Bar graph shows the results from ELISA for Rac activity (unpaired t -test, ** p = 0.002; n = 4 Cyfip1 +/– and 6 Wt ). (B) Western blot shows Rac1 levels in total hippocampal lysates from the same preparations used for the data in ( A ; statistics in the text). (C) Western blots for total and phosphorylated cofilin in lysates from the hippocampus (statistics in the text). (D) Western blots of WAVE1 from cell lysates in response to treatments indicated. (E) Blots of WAVE1 and PP2Ac a/b in hippocampal lysates from Wt and Cyfip1 +/– mice. (F) Graph of data in (E) showing the ratio of upper “phospho” to total WAVE1 (left y axis) and PP2Ac intensity (right y axis) (unpaired t -tests; pWAVE/WAVE1; * p = 0.002; PP2Ac, * p = 0.04, n = 3 each). (G) Phosphatase activity assay and (H) mean comparison of max OD at 405 nm, ** p = 0.003, n = 6 each.
Article Snippet: Primary antibodies included: mouse anti-GluA1 (NeuroMAB; 1:10 IHC), rabbit anti-GluA1 (Cell Signaling Technologies; 13185, 1:1,000), mouse anti-GluA2 (NeuroMAB; 1:10 IHC), mouse anti-GluA2 (MAB1189; 1:500), chicken anti-Homer (SySy; 160006, 1:500), mouse anti-SynGAP (Thermo Fisher Scientific; PA1-046, 1:500 IHC, 1:5,000 WB), mouse anti-PSD95 (Thermo Fisher; MA1-045, 1:1,000 WB), rabbit anti-Synaptophysin (PA1-1043, 1:5,000 WB), mouse anti-Cdk5 (Invitrogen; AHZ0492, 1:500), mouse anti-WAVE1 (mAb K91/361; NeuroMab), goat anti-PPA2c (Santa Cruz; sc-6110), rabbit anti-CaMKIIα (Abcam; EP1829Y, 1:1,000), rabbit-anti-p-Synapsin1-Ser 551 (Abcam; Ab32532), rabbit anti-p35 (Cell Signaling; 2680, 1:250), mouse anti-GAPDH (Millipore; MAB374, 1:4,000), and Phalloidin Alexa 647 (Thermo Fisher Scientific; A22287, 1:200).
Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Phosphatase Assay
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