Rabbit anti mcherry antibody

The primary antibodies used in the immunostaining experiments were as follows: mouse anti-GFP antibody (1:500, Roche, no. 11814460001), chicken anti-mCherry antibody (1:300, Novus Biologicals, no. NBP2-25158) and mouse anti-BRP antibody (1:100, Developmental Studies Hybridoma Bank, no. nc82). The secondary antibodies for immunostaining were anti-mouse labelled by Alexa 488 (1:1,000, ThermoFisher no. A28175) or Cy3 (1:1,000, Jackson ImmunoResearch Labs no. 115-165-146) and anti-chicken labelled by Alexa 647 (1:1,000, Jackson ImmunoResearch Labs, no. 103-605-155). The primary antibodies used in the western blots were mouse anti-GFP antibody (1:2,000, Roche, no. 11814460001), rabbit anti-mCherry antibody (1:2,000, Abcam, no. ab167453), rabbit anti-Ref2P antibody (1:500, Abcam, no. ab178440), rabbit anti-TMEM63A antibody (1:200, Novus Biologicals, no. NBP2-57359), mouse anti-GAPDH antibody (1:2,000, Proteintech, no. 60004-1-Ig) and mouse anti-tubulin antibody (1:2,000, Sigma, no. T9026). The secondary antibodies for the western blots were anti-mouse HRP antibody (1:4,000, Jackson ImmunoResearch Labs, no. 115-035-146) and anti-rabbit HRP antibody (1:4,000, Jackson ImmunoResearch Labs, no. 111-035-144).

For western blotting, 5 μg of total protein from either neurites or soma was separated on a 12.5% Laemmli PAAG, and proteins were transferred to the PVDF membrane. The membrane was probed with the following primary antibodies: rabbit anti-mCherry antibody 1:5000 (167453 Abcam), rabbit anti-Tuj1/TUBB3 1:5000 (T2200 Sigma), rabbit anti-Histone H3 1:5000 (ab1791 Abcam), rabbit anti-Homer 1:1000 (160003 Synaptic System). mChe-CDC42E7 levels were quantified with Fiji, normalized to the levels of TUBB3 and presented as the neurites/soma ratios.

Cells cultured after doxycycline induction were pelleted via centrifugation at 5000 r.p.m. and washed with PBS twice before protein extraction essentially as described previously^{20 (link)}. The fluorescent proteins GFPmut2 and mCherry were detected with mouse anti-GFP antibody (11814460001, Roche, Germany) and rabbit anti-mCherry antibody (5993–100, BioVision, USA), respectively, according to the manufacturers’ instructions. β-Actin was used as a loading control and detected with rabbit anti-β-actin antibodies (GTX109639, GeneTex, USA). The PageRuler Prestained Protein Ladder (26616, Thermo Fisher Scientific, USA) was used to indicate the relevant sizes of the protein targets. Signals from the detected proteins were obtained with SuperSignal West Pico Chemiluminescent Substrate (34080, Thermo Fisher Scientific, USA), and images were captured with an ImageQuant LAS 4000 Mini system (GE Healthcare Life Sciences). To quantify the levels of GFPmut2 and mCherry on the blots, images were analysed in triplicate with ImageJ (National Institutes of Health, USA), and then the quantified GFPmut2 levels were normalized to those of β-actin to determine the fold change in expression. Differential expression data were assessed by one-way ANOVA with Bonferroni’s comparison test, and the data were plotted as bar charts by GraphPad Prism 6 (GraphPad Software, USA).

Cells were cultured on gelatin coated glass coverslips, fixed in acetone:methanol (1:1) and stored at −20 °C. Slides were washed in PBS, before blocking in 5% (w/v) bovine serum albumin (BSA) in Hanks buffered salts solution (Gibco BRL, NY). Cells were stained using a rabbit anti-mCherry antibody (BioVision, CA) together with a goat anti-rabbit IgG-Alexa 555, secondary antibody (Molecular probes, CA). Bodipy 493/503 (Invitrogen) was prepared as a stock solution of 1 mg/ml in ethanol and used at a 1:1000 concentration PBS. Florescence was visualised using a Nikon TiE inverted microscope (Nikon, Tokyo, Japan).