Journal: EMBO Reports

Article Title: Cytolinker Gas2L1 regulates axon morphology through microtubule‐modulated actin stabilization

doi: 10.15252/embr.201947732

Figure Lengend Snippet: A, B Representative melt curves of various samples amplified with Gas2L1 primers (A) and amplification curves of GAPDH or Gas2L1 cDNA amplified in control and shRNA samples (B) from qPCR experiments shown in Fig A. cDNA samples of control (empty shRNA vector, in duplo ) and Gas2L1‐shRNA‐expressing neurons (G2L1 shRNA, in duplo ) show one peak in the melt curve (A), demonstrating specific amplification of Gas2L1 cDNA; negative water control (MQ instead of cDNA template) shows no amplification with Gas2L1‐specific primers; negative RNA control (RNA instead of cDNA as template) reveals minor unspecific product that is not amplified and therefore not interfering in the presence of cDNA. C Fine details of axon morphology of a DIV3 neuron overexpressing HA‐Gas2L1 as described in Fig B. Boxed regions (1 and 2) are enlarged below. Red asterisk indicates the soma. D Example of axon tracings for morphology analysis, using the DIV3 neuron expressing scrambled shRNA as shown in Fig B as a template. Boxed region is enlarged to the right. Blue tracing denotes the primary axon; the longest possible uninterrupted tracing from the soma to the tip of an axon branch. Red tracings denote non‐primary branches and black circle marks the position of the soma. E–G Rescue experiments showing the total axon length (E), primary axon length (F) and average axon branch length (G) in neurons co‐expressing scrambled (Scr) or Gas2L1 shRNA with GFP, or Gas2L1 shRNA with GFP‐Gas2L1, and HA‐β‐galactosidase from DIV0 to DIV3. Data belong to the experiment shown in Fig H and I. n = 34–43 neurons per condition (36 for Scr shRNA, 34 for G2L1 shRNA, 43 for G2L1 shRNA + GFP‐G2L1) from two independent experiments. H, I DIV3 neurons overexpressing GFP‐G2L1 stained for cortactin (H) or p34‐Arc (I), as well as merged images showing co‐localization between GFP‐Gas2L1 and cortactin (H) or p34‐Arc (I) (left panels). Boxes indicate zoomed regions (right panels). Data information: Scale bars: 25 μm in (C), 30 μm in (D, H, I). Data are displayed as means ± SEM. Mann–Whitney test, ns: not significant, *** P < 0.001.

Article Snippet: To create GW1‐GFP/HA‐ABD and GW1‐GFP/HA‐ABD‐Tail, α‐actinin CH domains were amplified from Gallus gallus cDNA (amino acids 35–246, NCBI ref. NM_204127.1) using a standard PCR‐based strategy.

Techniques: Amplification, shRNA, Plasmid Preparation, Expressing, Staining, MANN-WHITNEY