Antimouse ir800

Flow cytometry and quantitative Western analysis were performed as previously described.^{20 (link)} Antibodies used in flow cytometry were the following: Tuj1 (Covance, 1:2500), MAP2 (BD Biosciences, Franklin Lakes, NJ; 1:40), glial fibrillary acidic protein (GFAP; Millipore, 1:20), Tra1–81 (Millipore, 1:500), and SSEA3 (Millipore, 1:500). Secondary antibodies were antimouse Alexa 488 (1:1,000), antirat Alexa 488 (1:1,000), or antirabbit Alexa 647 (1:1,000). The following antibodies were used in quantitative Western analysis: frataxin (MitoSciences, Eugene, OR; 1:1,000), RPL13a (Cell Signaling Technology, Danvers, MA; 1:2,000), and RNA polymerase II (Millipore; 1:2,000). Antibodies against acetylated residues of histone H3 and H4 have been described.^{22 (link)} The following secondary antibodies were all obtained from LI-COR Biosciences (Lincoln, NE) and used at the same dilution (1:5,000): antimouse IR680, antimouse IR800, antirabbit IR680, and antirabbit IR800.

The protein sample (15 μL) was loaded on 6–12% Tris–glycine SDS-PAGE gels and run on a Mini-PROTEAN® BioRad gel system. Gels were transferred with the Invitrogen iBlot. Membranes were stained with Ponceau stain to verify transfer and equal protein loading and blocked with 3% BSA + 1 × TBST for 1 h at 24 °C. Primary antibodies and the following dilutions were incubated with the membranes for 12 h: anti-HA (1:1000; Cell Signaling; 3724S), anti-O-GlcNAc RL2 (1:1000; Abcam; ab2739), and anti-GAPDH (1:1000; Cell Signaling; D4C6R). The membranes were washed 3 × 5 min each wash with 1 × TBST and incubated with the following secondary antibodies and dilutions: anti-rabbit HRP (1:10,000; Rockland; 611–1302) and anti-mouse IR 800 (1:10,000; LI-COR; 925–32,210). The membranes were washed 3 × 5 min each wash with 1 × TBST, and results were obtained by chemiluminescence or IR imaging using Azure c600. The membranes were quantified using LI-COR Image Studio Lite, and graphs were made with GraphPad Prism 9.

Gel electrophoresis was performed using Novex NuPAGE 12% Bis-Tris gel in sodium dodecyl sulfate (SDS) MOPS running buffer (Invitrogen) at 200 V for 1 h. The gel was then transferred onto a nitrocellulose membrane using the TransBlot Turbo Transfer System (Bio-Rad, Hercules, CA), blocked with 5% nonfat dried milk, and stained with primary and subsequently secondary antibodies. Mouse anti-SNAP25 at 1:4000 dilution (Synaptic Systems, Gottingen, Germany) and mouse anti-β-actin at 1:10,000 dilution (Sigma) were used for the primary antibodies. Anti-mouse IR800 (Li-Cor) at 1:10,000 dilution was used as the secondary antibody. An Odyssey Infrared Imager (Li-Cor) was used to scan blots, and the relative amount of SNAP25 cleavage was quantified using ImageJ (National Institutes of Health, Bethesda, MD).