Anti tiar

After 24 h of transfection with DsRed-monomer-DGKη1 and/or EGFP-ASK3, COS-7 cells were incubated in DMEM with or without 500 mM sorbitol for 30 min. The cells were fixed in 3.7% paraformaldehyde. The coverslips were mounted using Vectashield (Vector Laboratories, Burlingame, CA, USA). Fluorescence imaging was performed using an Olympus FV1000-D (IX81) confocal laser scanning microscope (Olympus, Tokyo, Japan). Images were acquired using FV-10 ASW software (Olympus).
To observe stress granule markers, Ras GTPase-activating protein SH3-domain-binding protein (G3BP1) and T-cell intracellular antigen 1 related protein (TIAR), cells were fixed and then permeabilized in phosphate-buffered saline containing 0.1% Triton X-100 and 1% bovine serum albumin. Coverslips were incubated with an anti-G3BP1 (Cat. #: 611126, BD Biosciences, Franklin Lakes, NJ, USA) or anti-TIAR (Cat. #: 610352, BD Biosciences) mouse monoclonal antibody for 1 h and then incubated with Alexa 594-conjugated anti-mouse IgG (Molecular Probe) for 1 h.

Sources of commercial antibodies were as follows: anti-FUS (Santa Cruz, Dallas, TX, USA; 4H11), anti-Pur-alpha (Abcam, Cambridge, UK; ab77734), anti-Pur-alpha (Abcam; ab79936), anti-HA (Santa Cruz; Y-11), anti-HA (Santa Cruz; F-7), anti-Flag (Sigma-Aldrich, St. Louis, MO, USA; M2 and M2 affinity gel), anti-Flag fluorescein isothiocyanate (FITC) conjugated (Sigma-Aldrich; M2), anti-Phospho-eIF2-alpha (Cell Signaling Technology, Beverly, MA, USA; D9G8), anti-eIF2-alpha (Cell Signaling Technology; D7D3), anti-cleaved caspase-3 (Cell Signaling Technology; Asp175), anti-NeuN (Merck Millipore, Billerica, MA, USA; A60), anti-TIAR (BD Biosciences, Erembodegem, Belgium), anti-GAPDH (Chemicon-Merck Millipore), anti-puromycin 3RH11 monoclonal antibody (Kerafast, Boston, MA, USA), anti-FUS (Abcam; ab84078), anti-Islet-1/2 (DSHB, Iowa City, IA, USA; 39.4D5). Mouse monoclonal antibody specific for ribosomal protein S19 were prepared in our laboratory.^{31 (link)} FITC, Rhodamine, and aminomethylcoumarin-conjugated affinity-purified secondary anti'bodies, selected for absent cross-reaction, were from Jackson ImmunoResearch (West Grove, PA, USA).

The human VMRC-LCD cells, LCD cells stably expressing GFP/GFP-TDRD3 and MDA-MB-231 cells expressing Tet-on-sh TDRD3 have been described before [13 (link), 14 (link)]. Wild-type and Tdrd3-knockout MEF cells were generated from E12.5 mouse embryos following a standard MEF-generation protocol and the primary MEFs were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS. The HeLa, HEK293 and MDA-MB-231 cell lines were obtained from ATCC. All of the cell lines were maintained in DMEM containing 10% fetal bovine serum. The anti-TDRD3 antibody has been described before [13 (link)]. The anti-TDRD3 (CST) (cat#5942), anti-USP9X (cat#14898), anti-ADMA (cat#13522) and anti-SDMA (cat#13222) antibodies were purchased from Cell Signaling Technology. Anti-MCL-1 (cat# sc-819) was from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-ACTIN (cat# A5316) was from Sigma Aldrich (St. Louis, MO, USA). Anti-PRMT1 (cat# A300722A) was from Bethyl Laboratories (Montgomery, TX, USA). Anti-TIAR (cat# 610352) and anti-G3BP (cat# 61126) were from BD Biosciences (San Jose, CA, USA).