Ab104238

Cells were lysed in IP lysis buffer (Pierce/ThermoFisher Scientific) containing a protease inhibitor cocktail (Sigma-Aldrich). Total lysates were incubated with anti-FLAG (14793S; Cell Signaling Technology) or anti-IgG (ab172730; Abcam) overnight at 4 °C and then mixed with Protein A/G magnetic beads (Thermo Fisher Scientific) for 2 h at room temperature. Proteins were eluted and run on SDS-PAGE for western blot analysis. The following primary antibodies were used: anti-NONO (ab70335; Abcam), anti-N-cadherin (13116S; Cell Signaling Technology), anti-CD44 (3570S; Cell Signaling Technology), anti-BCL2 (4223S; Cell Signaling Technology), anti-BAX (50599-1-Ig; ProteinTech; Wuhan, China), anti-GPX1 (ab22604; Abcam), anti-CCN1 (14479S; Cell Signaling Technology), anti-SFPQ (15585-1-AP; ProteinTech), anti-PSPC1 (ab104238; Abcam), and anti-ZEB1 (3396S; Cell Signaling Technology).

Cells were lysed in ice-cold whole cell extract buffer B [50 mM Tris–HCl (pH 8.0), 4 M urea, and 1% Triton X-100] supplemented with complete protease inhibitor cocktail (Roche). The cell extracts were resolved with SDS-PAGE and analyzed with western blotting. The protein bands were visualized with ECL Blotting Detection Reagents. The antibodies used for western blotting included an anti-P54nrb antibody (Abcam, ab70335), anti-PSPC1 antibody (Abcam, ab104238), anti-SFPQ antibody (Abcam, ab11825), anti-STAT3 antibody (CST, 124H6), anti-pSTAT3Y705 antibody (Abcam, ab76315), anti-HSV-1 ICP0 antibody (Abcam, ab6513), anti-HSV-1 TK antibody (Santa Cruz Biotechnology, sc-28037), and anti-β-actin antibody (Proteintech, 60008-1-Ig).

HEK293T cells were grown on a glass slide and fixed in 4% formaldehyde in PBS. Cells were washed once with PBS, permeabilized with Triton X-100, dehydrated through a series of ethanol washes and hybridized overnight with a digoxigenin-labeled NEAT1 probe (Table S1). RNA FISH signal was detected by incubating with Alexa Flour 647-labeled anti-digoxygenin antibody (Roche) and examined on a Leica TCS SP8 STED microscope.
Immunofluorescence was carried out as described previously (Deng et al., 2019 (link)). Briefly, cells growing on glass slides were rinsed with PBS and fixed with 4% PFA for 15 min, and then permeabilized in 0.2% Triton X-100 followed by 30 min 5% BSA blocking. The cells were then stained with human anti-PSPC1 (Abcam, ab104238) or anti-SFPQ antibodies (Abcam, ab11825) in blocking buffer for overnight at 4 °C and washed three times with PBS. Then stained with Alexa Flour 647-labeled secondary antibodies (Jackson Laboratory) in blocking buffer for 1 h. After three washes with PBS, coverslips were mounted with VECTASHIELD mounting medium containing 0.5 μg/mL DAPI and then visualized at 100× on a CCD camera mounted on a Leica TCS SP8 STED microscope using imaging software. The magenta signal was defined as green by ImageJ (National Institutes of Health) before further colocalization analysis.