Journal: Journal of neurochemistry

Article Title: Neuronal filopodium formation induced by the membrane glycoprotein M6a (Gpm6a) is facilitated by coronin-1a, Rac1, and p21-activated kinase 1 (Pak1).

doi: 10.1111/jnc.13552

Figure Lengend Snippet: Fig. 1 (a) Gpm6a and Rac1 co-immunoprecipitate with Coro1a from rat hippocampal lysates. Western blot of proteins co-immunoprecipi- tated from rat hippocampal lysates using anti-Coro1a antibody and probed with anti-Gpm6a, anti-Rac1 and anti-Coro1a antibodies. For this purpose, the membrane (different kDa areas) was cut and incubated with indicated antibodies. Non-immune rabbit serum was used as a control. Gpm6a and Rac1 are present in the anti-Coro1a immunoprecipitate. Bands representing Gpm6a are indicated by stars. (b) Coro1a co-localizes with Gpm6a in hippocampal neurons. Confocal image of hippocampal neurons (4 DIV) co-immunostained with anti- bodies against Gpm6a (red), Coro1a (green) and dendritic marker MAP2 (blue). A portion of Gpm6a-labeled spots co-localizes with Coro1a (arrowheads; insets 1 and 2). Scale bar, 20 lm.

Article Snippet: Mammalian expression plasmids: pRFP-C1 encoding the red fluorescent protein (RFP), pEGFP-C1 (Clontech Laboratories, © 2016 International Society for Neurochemistry, J. Neurochem. (2016) 137, 46--61 Mountain View, CA, USA) encoding the enhanced green fluorescent protein (EGFP), RFP-tagged wild-type (wt) Gpm6a (Gpm6aRFP) described previously (Alfonso et al. 2005), EGFP-tagged wt Coro1a (wtCoro1a-EGFP), and a deletion mutant containing only 5 WD repeats (aa 65-306) of Coro1a [Coro1a(WD1-5)-EGFP] kindly provided by Dr William Trimble (Yan et al. 2005), EGFPtagged Rac1 T17N [Rac1DN-EGFP; Addgene#12982 (Subauste et al. 2000)], EGFP-tagged Rac1 Q61L [Rac1CA-EGFP; Addgene#12968 (Subauste et al. 2000)], myc-tagged Pak1 K299R [Addgene#12210 (Sells et al. 1997)], and myc-tagged Pak1 H83L/H86L [Addgene#12211 (Sells et al. 1997)]. siRNAs: siGENOME non-targeting siRNA Pool #2, Coro1a siRNAs targeting either its coding DNA sequence (CDS) [prevalidated in Suo et al. (Suo et al. 2014)] or its 30-UTR region (custom designed).

Techniques: Western Blot, Membrane, Incubation, Control, Marker, Labeling