Mouse anti alpha tubulin

All primers used for cloning projects are listed in Table 1. The full-length coding sequence of HirFAAH2 was generated from total RNA using SuperScript III One-Step RT-PCR system with Platinum Taq DNA Polymerase (Thermo Fisher Scientific Inc., Rockford, IL). To generate an expression construct with 3’ eGFP tag the HirFAAH was inserted into pcDNA3.1eGFP (Addgene plasmid #13031; http://n2t.net/addgene:13031;RRID:Addgene_13031). Single point mutation was generated using the GeneTailor Site-Directed Mutagenesis System (Thermo Scientific). All constructs were confirmed by sequencing (Eurofins MWG Operon, Huntsville, AL). Plasmids were transfected into 293HEK cells (ATCC, Manassas, VA) using Lipofectamine 3000 (Thermo Scientific) as previously described by the manufacturer. For Western blots equal amounts of protein were separated on 10% SDS-PAGE gels and transferred to PVDF. Primary antibodies used were mouse anti-alpha tubulin (Abcam, Cambridge, MA), and mouse anti-GFP (B-2) (Santa Cruz Biotechnology, Dallas, TX). Goat anti-mouse IRDye 680LT was used as a secondary antibody (LiCor Biosciences, Lincoln, NE). Images were taken using the Odyssey CLx and processed using the Image Studio version 5.2 (LiCor Biosciences).

Protein samples were mixed with SDS sample buffer and boiled at 100 °C for 10 min, and then loaded to 10% acrylamide gels for SDS-PAGE. Proteins were transferred to nitrocellulose membranes in buffer containing 20% MeOH. Membranes were blocked with 5% skim-milk (BD) or 5% BSA for 1 h, followed by overnight incubation at 4 °C with primary antibodies: mouse anti human TDP-43 (Proteintech, 1:4000), rabbit anti TDP-43 (Proteintech, 1:2000), rabbit anti ERK1/2 (tERK; Sigma, 1:10,000), mouse anti alpha-tubulin (Abcam, 1:5,000), rabbit anti MAP2 (Milipore, 1:1000), mouse anti TAU-5 (Abcam, 1:250). Membranes were then washed with TBST and incubated 2 h at RT with secondary HRP antibody (Donkey anti rabbit and donkey anti mouse, Jackson Laboratories, 1:10,000), or HRP-Goat-IgG2a-anti-mouse (Jackson, 1:20,000; for puromycin blots) washed with TBST and visualized in iBright 1500 ECL imager (Life Technologies) after 5 min incubation with ECL reagents. Quantification was performed using FIJI ImageJ V.2.0.0 software.

Cell extracts and precipitated supernatants were analyzed by immunoblot using goat anti-IL-1β (R&D), mouse anti-NLRP3 (Cryo-2), rabbit anti-ASC (AL177), mouse anti-Caspase-1 (Casper-1) (all three from Adipogen), and mouse anti-alpha-tubulin (Abcam).

Immunoblot analysis was performed as described in [13 (link)]. Mouse anti-RNase L antibody 2E9 (Novus Biologicals catalog no. NB100-351) was used at 1:1500. Histone H3 antibody (Thermo Fisher Scientific; NB500-171) was used at 1:1000. Mouse anti-alpha tubulin (Abcam: ab18251) was used at 1:1000. Rabbit anti-GAPDH (Cell Signaling Technology: 2118L) was used at 1:2000. Anti-rabbit immunoglobulin G (IgG), horseradish peroxidase (HRP)–linked antibody (Cell Signaling Technology: 7074S) was used at 1:3000. Anti-mouse IgG, HRP-linked antibody (Cell Signaling Technology: 7076S) was used at 1:10,000 Crude nuclear and cytoplasmic fractionation was performed as described in [58 (link)].

Oocytes were exposed to acidic tyrode solution (pH 2.5) for a few seconds to remove the zona pellucida followed by three washes in M2 medium. Oocytes were then fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) in phosphate-buffered saline (PBS) at room temperature for 30 min, followed by permeabilization in PBS containing 0.5% Triton X-100 for 2 h at room temperature. Sample blocking was conducted with 1% bovine serum albumin (BSA, Amresco, Solon, OH, USA) in PBS containing 1/1000 Tween-20 (Amresco) and 1/10,000 Triton X-100. After blocking, samples were incubated with primary antibodies overnight at 4°C. For primary antibodies, we used mouse anti-alpha tubulin (1:2000, Abcam, Cambridge, UK) and anti-gamma tubulin (1:500, Abcam). For secondary antibodies, we used DyLight 549-conjugated donkey anti-mouse (1:100, Jackson ImmunoResearch Laboratories, West Grove, PA, USA). DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI, 5 μg/mL, Roche, Mannheim, Germany) for 10 min. After staining and washing, samples were mounted on glass slides using Vectashield mounting medium (Vector Labs, Burlingame, CA, USA) and examined with a confocal laser-scanning microscope (Nikon, A1R, Tokyo, Japan). Images were analyzed with NIS-Element AR 3.0 software. Chromosome spreading analysis was performed as we described previously [63 (link)].

The following antibodies were used: mouse anti-MiTF (Abcam), mouse anti-FANCD2 (Santa Cruz), rabbit anti-FANCD2 (Abcam), rabbit anti-FANCA (Bethyl), mouse anti-Flag (Sigma), rabbit anti-p53 serine 15 (Cell Signaling Technology), mouse anti-p53 (Santa Cruz), rabbit anti p27 (Cell Signaling), rabbit anti-p21 (Santa Cruz), mouse anti-Cyclin-A (Abcam), rabbit anti-53BP1 (Abcam), mouse anti-gH2AX (Millipore), mouse anti-alpha-tubulin (Abcam), mouse anti-vinculin (Abcam), and goat anti-actin (Abcam).