Anti myc tag 9b11

The following antibodies and working concentration were used in this study: anti-CTTNBP2 (A5, A7, and 9W, rabbit, homemade, 0.5 μg/ml) [5 (link), 20 (link)], anti-Myc tag (9B11, Cell Signaling Technology, 1/1000 for staining; 06-549, Millipore, 1 μg/ml), anti-HA tag (3F10, Roche, 0.5 μg/ml), anti-HA tag (Y-11, Santa Cruz Biotechnology, 0.5 μg/ml), anti-GFP (ab13970, Abcam, 0.5 μg/ml), anti-αtubulin (B-5-1-2, Sigma-Aldrich, 1 μg/ml), anti-acetyl tubulin (6-11B-1, Sigma-Aldrich, 1 μg/ml), anti-βactin (AC-74, Sigma-Aldrich, 1/1000), anti-cortactin (H-191, Santa Cruz Biotechnology, 0.5 μg/ml), anti-FOS (#2250, clone 9F6, Cell Signaling Technology, 1/200), anti-mouse HRP (NA931, GE Healthcare, 1/5000), anti-rabbit HRP (NA934, GE Healthcare, 1/5000), anti-chicken Alexa Fluor 488 (A-11039, Invitrogen, 1 μg/ml), anti-mouse Alexa Fluor 555 (A-21424, Invitrogen, 1 μg/ml), anti-rat Alexa Fluor 594 (A-21209, Invitrogen, 1 μg/ml), and anti-rabbit Alexa Fluor 647 (A-21244, Invitrogen, 2 μg/ml).

An aliquot of the total lysates was used for immunoblotting. 0.1% SDS was added to the lysates, followed by sonication (3 cycles, 10 s each). Samples were run on 4–20% Criterion Tris-HCl gels (Bio-Rad Laboratories) and electrophoretically transferred to a nitrocellulose membrane (Thermo Fisher Scientific). Residual protein-binding sites were blocked by incubation with 5% non-fat milk in 1XTBST (1X TBS plus 0.1% Tween 20) 1 h at RT, followed by an O/N incubation at 4 °C with primary antibodies diluted in 20% SuperBlock buffer (Thermo Fisher Scientific) in 1XTBST. Mouse anti-N-Cadherin A60 (BD, Franklin Lakes, NJ), mouse anti-actin Ab5 (BD) were diluted 1:5000; anti-Myc-tag 9B11 (Cell Signaling, Danvers, MA) was diluted 1:1000. Appropriate isotypes secondary antibodies HRP-conjugated were diluted 1:2000 in 5% non-fat milk 1XTBST, and added for 1 h at RT. Every step was followed by 3 or 4 washes in 1X TBST. Detection was performed using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) or SuperSignal West Dura extended duration substrate (Thermo Fisher Scientific) and exposed to x-ray films.

Antibodies used include UCH-L1 (3524), raptor (2280), rictor (9476), 4EBP1 (2845), p4EBP1^T70 (5078), p4EBP1^T37/46 (2855), p4EBP1^S65 (9451), pS6K (9208), S6K (49D7), AKT (4691), pAKT^S473 (4060), eIF4A (C32B4), eIF4E (C46H6), anti-DYKDDDDK Tag (8146), anti-MYC tag (9B11) from Cell Signaling Inc. (Danvers, MA, USA), anti-HA (3F10, Roche Applied Science, Indianapolis, IN, USA) tubulin (T9026) from Sigma, mTOR (A301-143a), PHLPP (A300-660A) from Bethyl Laboratories (Montgomery, TX, USA), eIF4G (ab31217) from Abcam. Immunoprecipitations were performed as described using lysates prepared with 0.3% CHAPS (mTOR/rictor/raptor)^{4 (link),30 (link)}, 0.5% NP40 (BioID2-UCHL1, UCHL1-HA) or 1% SDS (raptor for detection of FLAG-Ub)^{4 (link)}. Immobilized m⁷GTP (AC-155) was from Jena Bioscience (Jena, Germany). M⁷GTP pulldowns were performed as described^{3 (link)}.

Cells were harvested in RIPA (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% vol/vol Igepal CA-630, 6 mM sodium deoxycholate, 1 mM EDTA) with a protease inhibitor cocktail (Sigma). Western blotting was performed as described previously (31 (link)). Proteins (20 μg) were separated by SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) using 12% acrylamide gels. Primary antibodies used were rabbit polyclonal anti-NIS (1:1000; Proteintech, Rosemont, IL), mouse monoclonal anti-MYC-Tag 9B11 (1:1000; Cell Signaling Technology), rabbit monoclonal anti-HA Y-11 (1:1000; Santa Cruz), and mouse monoclonal β-actin AC-15 (1:10,000; Sigma).

Antibodies for immunoblotting were from commercially available sources. anti-δ-catenin (#611537, BD Bioscience); anti-GFP (#G1544, Sigma); anti-p-Erk1/2 (#16982, Santa Cruz); anti-Erk1 (SC-94, Santa Cruz); anti-EGFR 1005 (#03, Santa Cruz); anti-EGFR (D20) (#31156, Santa Cruz) anti-E-cadherin (SC-7870, Santa Cruz); anti-py20 (SC-508, Santa Cruz) and anti-Myc-Tag (9B11) (#2276,Cell signaling).
Antibodies for immunofluorescence were also obtained commercially: anti-E-cadherin (#3195, Cell signaling), anti-EGFR 225 (MA5-12880, ThermoFisher Scientific).
Control siRNA and siRNA anti-δ-catenin were purchased from Sigma-Aldrich.

Immunofluorescence staining was conducted as described previously (30 (link)). Primary antibodies used were mouse monoclonal anti-MYC-Tag 9B11 (1:750; Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-HA Y-11 (1:100; Santa Cruz, Dallas, TX), mouse monoclonal anti-HA 16B12 (1:100; BioLegend, San Diego, CA), rabbit monoclonal anti-Na⁺/K⁺/ATPase (Alexa Fluor^® 488), EP1845Y (1:50; Abcam, Cambridge, United Kingdom), and rabbit monoclonal anti-Na⁺/K⁺/ATPase EP1845Y (1:250; Abcam).
A Zeiss LSM 510 confocal microscope with × 40 objective was used to perform confocal microscopy (Carl Zeiss AG, Oberkochen, Germany). Epifluorescent microscopy was performed using × 40 objective on a Leica DM6000 fluorescent microscope (Leica Microsystems, Wetzlar, Germany). The Duolink In Situ Fluorescence Protocol with Detection Reagents Red Kit (Sigma) was used as per the manufacturer's instructions. Cells were transfected for 48 hours before fixation, permeabilization, and addition of anti-MYC and anti-HA antibodies.

WT pET23a-Trx-hnRNPK was prepared according to a previous study [26 (link)]. The cDNA segment of the KI region (hnRNPK amino acid sequence: 240–336) was subcloned into the expression vector pET23a-Trx by KpnI/XhoI digestion. Full-length cDNA DDX3 and its truncated variants were inserted into the PGEX-5X-1 vector. In addition, full-length cDNAs encoding human DDX3 and C-terminal truncated DDX3 were inserted into a pCDNA4-myc/His vector. The cDNA fragment of hnRNPK or hnRNPK-ΔKI was subcloned into the expression vector of pCDNA3-N-terminal Venus through KpnI/EcoRI digestion.
The antibodies used in the present study were purchased from vendors as indicated: Anti-DDX3 (#54169) from Arigo Biolaboratories (Hsinchu, Taiwan); anti-JUND (#102135) and anti-GAPDH (#100118) were from GeneTex lnc (San Antonio, TX, USA); Anti-β-actin (AC-15), anti-hnRNPK/J (3C2), and anti-Flag (M2) were from Sigma-Aldrich (St Louis, MO, USA); and anti-Myc tag (#9B11) and anti-cleaved caspase-3 (#9661) were from Cell Signaling Technology (Beverly, MA, USA).