Ab290

The following primary antibodies were used for this study: rabbit anti‐VAPA (homemade #1006‐04; Teuling et al, 2007) and anti‐VAPB (homemade #1006‐00; Teuling et al, 2007); mouse anti‐synaptotagmin (SySy, 105311, clone 604.2); rabbit anti‐SCRN1 (SySy, 289003; used in Fig 1E), rabbit anti‐SCRN1 (Abcam, ab105355; used in Fig EV1D), and rabbit anti‐SCRN1 (Sigma, HPA024517, RRID:AB_2184811; used for all other experiments; validation SCRN1 antibodies in Fig EV1G–J); guinea pig anti‐vGlut (Millipore, ab5095); rat anti‐HA (Roche, 1867423; used for immunostainings); mouse anti‐HA (BioLegend, mms‐101p, clone 16B12; used for immunoblots); mouse anti‐actin (Chemicon, MAB1501R, clone C4); rabbit anti‐GFP (Abcam, ab290); mouse anti‐Myc (Santa Cruz, SC40, clone 9E10); and mouse α‐Tubulin (Sigma, T5168, clone B‐5‐1‐2, RRID:AB_477579). The following secondary antibodies were used for this study: anti‐rabbit Alexa 488 (Life Technologies, A11034), anti‐rabbit Alexa 568 (Life Technologies, A11036), anti‐rat Alexa 568 (Life Technologies, A11077), anti‐guinea pig Alexa 568 (Life Technologies, A11075), anti‐mouse Alexa 647 (Life Technologies, A21236), anti‐mouse anti‐HRP (Dako, P0260), anti‐rabbit anti‐HRP (Dako, P0399), anti‐mouse IRDye 680LT (Li‐Cor, 926‐68020), and anti‐rabbit IRDye 800CW (Li‐Cor, 926‐32211).

C. elegans strains were cultured as previously described by Brenner [11 (link)]. Strain VS20 hjIs67[Patgl-1::atgl-1::GFP] [12 (link)] was obtained from CGC and subsequently crossed into CB1370 daf-2(e1370) and MR1000 daf-2(e1370); aak-1(tm1944); aak-2(ok524) strains. Strains MR1348 daf-2(e1370); rrEx226[sur-5p::GFP::ATGL-1::HA; rol-6(gf)] and MR1413 daf-2(e1370); rrEx239[syr-5p::GFP::ATGL-1(S303A)::HA; rol-6(gf)] are described in [9 (link)]. The strain atgl-1(tm3116) harbors a 423bp deletion of the atgl-1 gene and was obtained from National BioResource Project, Tokyo, Japan. Rabbit polyclonal antibody against ATGL-1 was raised using a synthetic peptide CTKRKVPDEPTTSKR (GenScript). Anti-PAR-5 antibody was a gift from Dr. Andy Golden. Anti-GFP (abcam ab290), anti-ubiquitin (Santa Cruz SC8017) and anti-P-14-3-3 (Cell Signaling #2981S) antibodies are available commercially.

Total protein (50 μg) or histone (5 μg) were separated by SDS-PAGE and transferred to PVDF membrane(GE Healthcare). The membrane was blocked with 5% milk and probed with specific primary antibodies and secondary antibodies. The blots were developed with ECL Advance Western Blotting Detection Kit (Amersham, #34080). The antibodies used in this study include Anti-Nanog antibody (Bethyl, A300-397A), anti-Rad51 antibody (Abcam, Ab63801), anti-HA antibody (Bethyl, A190-108A), anti-γH2AX antibody (Bethyl, A300-081A), anti-H3 antibody (Abcam, ab1791), anti-GST antibody (Santa Cruz, sc-138), anti-GFP (Abcam, ab290), anti-β-tubulin (Santa Cruz, sc-166729) and anti-GAPDH antibody (Bethyl, A300-641A).

16Q-TBP and 59Q-TBP cells were induced using 6 µg/ml doxycycline (Clonetech) and 5 mM sodium butyrate (Sigma). Cells were harvested 48 h after treatment using TRIzol reagent (Invitrogen) for RNA isolation. RNA was isolated as per the manufacturer’s protocol. cDNA synthesis was performed using M-MuLV Reverse Transcriptase (NEB) and quantitative PCR (qPCR) was performed using KAPA SYBR Master Mix. Primers used in the study have been listed in Supplementary Table 1. For protein extraction, induced cells were harvested 48 hr after treatment using RIPA buffer (Sigma). Protein was quantitated using BCA method and Immunoblotting was performed. Primary antibodies for STAT1 (Cell Signaling Technologies, 9172), pSTAT1 (Cell Signaling Technologies, 7649), TBK1 (Abcam, ab40676) and IRF3 (Cell Signaling Technologies, 4302) were used at 1:1000 dilution. Anti-polyglutamine-expansion specific antibody (Millipore, MAB1574), Anti-GFP (Abcam, ab290) and GAPDH (Santa Cruz Biotechnology, sc32233) were used at 1:5000 dilution. was used a housekeeping control at 1:5000 dilution. Secondary antibodies used was conjugated with Horseradish Peroxidase or by infrared dye and hence, blots were developed using chemiluminescence or scanned in infrared LI-COR Odyssey scanner, respectively.

Embryos were embedded with optimum cutting temperature compound after fixation and were sectioned at 10–14 μm increments. After washing with PBS and 1 h of preincubation with 1% BSA in PBS for blocking, sections were incubated with 500 × diluted primary antibodies (anti-GFP: Nakalai GF090R or Abcam ab290; anti-Tbx1: Santa Cruz sc-17874; anti-Tbx18: Santa Cruz SC17869; anti-Wt1: Abcam ab10670; anti-Nkx2-5: Abcam ab35842; anti-TnT: Thermo MS-295-P0; anti-Hcn4: Alomone Labs APC052; anti-RFP: MBL PM005; anti-β-catenin: Millipore 05-665; anti-Ki67: Abcam ab15580) at 4 °C overnight. The sections were then incubated with 500 × diluted secondary antibodies conjugated with Alexa Fluor 488 or 555 (Molecular Probes). Nuclei were counterstained with 4,6-diamidino-2-phenylindole. For LacZ-staining, frozen sections were incubated with X-gal solution at room temperature overnight after washing with PBS.