Rabbit anti ddx6

The following antibodies were used: rabbit anti-eIF4E2 (4EHP) (Genetex, GTX103977), mouse anti-eIF4E (BD Biosciences, 610270), rabbit anti-eIF4ENIF1 (4E-T; abcam, ab55881), rabbit anti-DDX6 (Bethyl Laboratories, A300-460A), rabbit anti-CNOT1 (Proteintech, 14276–1-AP), mouse anti-α-Tubulin (Santa Cruz, sc-23948), mouse anti-β-actin (Sigma, A5441), mouse anti-Flag (Sigma, F3165), rabbit anti-HA (Sigma, H6908), mouse anti-V5 tag (Invitrogen, R960-25), rabbit anti-PARP (Cell Signaling Cat# 9532S), rabbit anti-DUSP6 (abcam Cat# ab76310), rabbit anti-DUSP7 (abcam Cat# ab100921),), rabbit anti-CNOT9 (RQCD1) (Proteintech Cat# 22503–1-AP), mouse GAPDH (Santa Cruz, sc-32233), rabbit anti-phospho-ERK1/2 (Thr202/Tyr204; Cell Signaling Cat#4370), mouse anti-MEK1/2 (Cell Signaling Cat# 4694S), rabbit anti-phospho-MEK1/2 (Ser217/221; Cell Signaling Cat# 9121S), rabbit anti-phospho-RPS6 (Ser240/244) (Cell Signaling Cat# 2215), and mouse anti-RPS6 (C-8).
The following siRNA and shRNAs were used: ON-TARGETplus Non-targeting Control Pool (Dharmacon, D-001810-10-05), 4EHP siRNA SMARTpool (Dharmacon, L-019870–01), eIF4ENIF1 (4E-T) siRNA SMARTpool (Dharmacon, L-013237–01), CNOT1 siRNA SMARTpool (Dharmacon, L-015369–01-0005), CNOT9 siRNA SMARTpool (Dharmacon, L-019972–00), Non-Targeting shRNA Controls (Sigma, SHC002), and EIF4E2 shRNA (Sigma, TRCN0000152006).

Ultrastructural studies were carried out on glutaraldehyde-fixed cells embedded in Epon™ 812 as described in Souquere et al.^{72 (link)}. Cells were fixed for 1 h at RT in 1.6% glutaraldehyde (Taab Laboratory Equipment, Reading, UK) in 0.1 M phosphate buffer pH 7.3. Then, cells were pelleted and dehydrated in increasing concentrations of ethanol and embedded in Epon. Polymerization was carried out for 48 h at 64 °C. Ultra-thin sections were collected on Formvar-carbon-coated copper grids (200 mesh).
Anti-Ferritin and anti-DDX6 immunogold labeling was performed on ultra-thin sections of glutaraldehyde-fixed and formaldehyde-fixed Lowicryl K4M-embedded cells, respectively. Embedding was at −20 °C in an AFS2 Freeze Substitution Processor apparatus (Leica Microsystems), polymerization under UV light was for 2 days at −20 °C, followed by 2 days at 20 °C. Electron microscopy grids were incubated with the primary antibody (rabbit anti-ferritin, Abcam 75973; rabbit anti-DDX6, Bethyl Laboratories) and subsequently with a goat anti-rabbit secondary antibody coupled to 10 nm gold particles (BB International).
Sections were analyzed with a FEI Tecnai transmission electron microscope and images were taken with a SIS MegaviewIII CCD camera.