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

Journal: Traffic (Copenhagen, Denmark)

Article Title: Inhibition of Autophagy Alters Intracellular Transport of APP Resulting in Increased APP Processing.

doi: 10.1111/tra.12934

Figure Lengend Snippet: FIGURE 6 | Reduced localization of RUSH mCherry-APP-EGFP to LAMP1-positive vesicles. (A) ATG9 WT (left) and KO (right) HeLa cells stably expressing the Str-KDEL ER hook and mCherry-APP-EGFP were fixed after 60, 120, and 180 min of treatment with 40 μM biotin and were immunostained for the lysosome marker LAMP1 (magenta). (B) Co-occurrence analysis of LAMP1 and EGFP (left) or mCherry (right) signal was performed over the whole cell using automated thresholding and the image calculator operator “AND” in Fiji to generate an image with only co- occurring pixels. (C) Colocalization analysis of mCherry-APP-EGFP and LAMP1-positive structures between 0.01 and 1 μm2 in size was performed using the “analyze particles” function in Fiji. (D) Confocal microscopy images as shown in (A) were analyzed for LAMP1 vesicular structures. The left graph displays the number of LAMP1 vesicular particles per μm2, and the size of those particles is depicted in the right graph at 60, 120, and 180 min after releasing mCherry-APP-EGFP from the ER. (E) Co-occurrence analysis of mCherry and EGFP signal was conducted as described in (B). Statistical analysis was carried out using Mann–Whitney test on data from three independent experiments with n > 130 cells per condition (significance levels: *p < 0.05; **p < 0.01; ***p < 0.001).

Article Snippet: Due to insufficient ER retention of mCherry- APP- EGFP by the Ii- Str hook, the lentiviral plasmid pCDH_Str- KDEL (from Franck Perez, Addgene plasmid #65307) was used as another ER hook in conjunction with the cloned pLenti_CMV_SBPmCherry- APP- EGFP_blas vector.

Techniques: Stable Transfection, Expressing, Marker, Confocal Microscopy, MANN-WHITNEY