Ab233

Samples were fixed in 4% PFA in PBS overnight at 4°C and then washed with 0.25% Triton X-100/PBS (PBTx). Brains were manually dissected and blocked for at least 1 h in 2% goat serum/2% DMSO/PBTx at room temperature or overnight at 4°C. Antibody incubations were performed in blocking solution overnight at 4°C using chicken anti-GFP (1:400, GFP-1020, Aves Labs), rabbit anti-TagRFP (1:100, AB233, Evrogen), or rabbit anti-dsRed (1:400, 643496, Takara Clontech) primary antibodies, and Alexa Fluor 488 and 568 secondary antibodies (1:500, Life Technologies). Samples were mounted in 50% glycerol/PBS and imaged using a Zeiss LSM 780 confocal microscope. Quantification of neurons (Figure 5—figure supplement 2M and Figure 6C) and c-fos intensity per NPVF neuron (Figure 5—figure supplement 2G) was performed blind prior to genotyping.

NaOH/TCA protein extracts [99 (link)] were prepared and subjected to SDS-PAGE. Resolved proteins were transferred to PROTRAN nitrocellulose membrane (Sigma-Aldrich/Merck, Darmstadt, Germany) or PVDF (Sigma-Aldrich/Merck, Darmstadt, Germany). Membranes were blocked with 5% non-fat milk (Regilait, Saint-Martin-Belle-Roche, France) and probed with corresponding antibodies. Red fluorescent protein fusions were detected with an anti-tRFP antibody (AB233, Evrogen, Moscow, Russia) that recognizes TagRFP-T and mTagBFP, and with an anti-RFP antibody (3F5, Chromotek, Planegg-Martinsried, Germany) recognizing mRFP and mCherry fluorescent proteins. The antibodies were used at dilution 1:1000 at 4 °C overnight. As a secondary antibody, the goat anti-rabbit IgG antibody conjugated to HRP (32460, Pierce-ThermoFisher Scientific, Waltham, MA, USA) and the goat anti-mouse IgG antibody conjugated to HRP (32430, Pierce-ThermoFisher Scientific, Waltham, MA, USA) were used both at dilution 1:600. GFP protein fusions were assayed with the GFP(B-2) HRP conjugate (sc-9996, Santa Cruz Biotechnology, Dallas, TX, USA) at dilution 1:1000, 1 h at RT or overnight at 4 °C. Proteins were detected by SuperSignal West Dura Substrate (Pierce-ThermoFisher Scientific, Waltham, MA, USA).

Cover-slips of 0.17 mm thickness (no. 1.5) were coated with poly-l-lysine (Sigma, P8920) for 30 min at 37°C, washed twice with MilliQ water and left to dry overnight at room temperature. Cryosections of 20 μm thickness were mounted directly on the cover-slips. The EMTB-GFP and F-tractin-mKate2 fluorescent signals were amplified with anti-GFP (Abcam, ab6673) and anti-tRFP (Evrogen, AB233; RRID:AB_2571743) primary antibodies (both at 1:300), respectively. Secondary antibodies were conjugated with Alexa 488 and Alexa 568 (ThermoFisher, A-11055, ThermoFisher, A-10042). Tissue sections were mounted on Slowfade Gold antifade (ThermoFisher, S36936) or Prolong Diamond (ThermoFisher, P36965) mounting media.

Wild type (WT) and NRVMs infected with TagBFP2, TRPC1-TagBFP2 or shRNA-TRPC1-TagBFP2 were lysed in RIPA buffer. Protein was collected and protein concentration was quantified with the Pierce BCA protein assay kit (23227, Thermo Fisher Scientific). Samples containing 2 μg/μl protein lysate with radio immunoprecipitation assay (RIPA) buffer, 2-Mercaptoethanol, and 6× loading dye were heated to 70°C for 10 min for protein reduction. 35 µg protein was loaded on a 4%–12% Bis-Tris Plus Gel (NW04120BOX, Thermo Fisher Scientific) and electrophoresed in MOPS running buffer (B001, Invitrogen) at 200 V for 35 min. Protein was transferred onto a 0.45 μm nitrocellulose membrane in Tris-Glycine-Methanol buffer for 1 h at 250 mA. The transferred blot was blocked for 1 h at room temperature in a solution of 5% BSA in Tris-Buffered Saline with Tween 20 (TBS-T). Primary antibodies for tRFP (AB233, Evrogen) and GAPDH (AB8245, Abcam) were applied at a dilution of 1:2,500 in blocking solution for 1.5 h. Secondary antibodies (AB97069 and AB97040, Abcam) and Precision Protein StrepTactin-HRP (1610380, Bio-Rad) were applied at 1:30,000 for 1 h. The blot was then incubated in Western Bright ECL HRP substrate kit (K012045, Advansta, San Jose, CA, United States) for 2 min and imaged on Bio-Rad Imager (Bio-Rad).

These studies used commercial mouse monoclonal antibodies against ALIX (sc-53540; Santa Cruz Biotechnology, Dallas, TX), α-tubulin (DM1A; Sigma, St. Louis, MO), lamin A/C (sc-7292; Santa Cruz Biotechnology); commercial rabbit polyclonal antibody against GFP (598; MBL, Nagoya, Japan), TagRFP (AB233; Evrogen, Moscow, Russia), CHMP4B (ab105767; Abcam, Cambridge, UK).