G3bp1

The following control siRNA and siRNAs used to suppress UBAP2L, G3BP1/2, FXR2, and NOP56/58 expression were purchased from Thermo Fisher Scientific (Waltham, MA USA): Silencer® Select siRNA Control (4390843); and Silencer® Select Pre-designed siRNA UBAP2L #1(s19176) and #2(s230223), G3BP1 #1 (s19754) and #2 (s19755), G3BP2 #1 (s19206) and #2 (s19207), FXR2 #1 (s18243) and #2 (s18244), NOP56 #1 (s20642) and #2 (s20643), and NOP58 #1 (s28390) and #2 (s28391). HeLa cells were transfected with 5 nM siRNA using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA. USA).

HeLa P4 cells^{82 (link)} were grown on poly-l-lysine coated 12 mm coverslips (No 1.5), permeabilized with 0.003–0.005% digitonin in TPB (20 mM HEPES pH 7.3–7.4, 110 mM KOAc, 2 mM Mg(OAC)₂, 1 mM EGTA, 2 mM DTT and 1 µg ml^–1 each aprotinin, pepstatin and leupeptin). After several stringent washes, nuclear pores were blocked by 15 min incubation with 200 µg ml^–1 wheat germ agglutinin (WGA) on ice. Cells were then incubated for 30 min at room temperature in the absence (background control) or presence of 400 nM recombinant/purified tau protein (K18, K25, hTau40, or Ac-hTau40) or 5 µM TMR-labeled peptides, respectively, in TPB. Tau proteins to be compared quantitatively had similar DOL ( ± 3%) or were corrected to achieve similar DOLs by mixing with unlabeled protein of the same concentration (“DOL corrected”). After several stringent washes to remove non-bound protein, cells were fixed and subjected to immunofluorescence for G3BP1 (Proteintech) and TIA-1 (Santa Cruz, C-20; sc-1751) using Alexa 555 (Thermo Fisher Scientific) and Alexa 647-labeled secondary antibodies (Thermo Fisher Scientific), respectively, to visualize SGs. Tia-1 was stained using an Alexa 647-labeled secondary antibody (Thermo Fisher Scientific), while G3BP1 was stained using either Alexa 488 (with TMR-labeled peptides) or Alexa 555 (with tau proteins) secondary antibodies (Thermo Fisher Scientific).