Ddx3x

Whole-cell extracts were prepared with 1% sodium dodecyl sulfate (SDS) lysis buffer with freshly added 1% proteinase inhibitor cocktail (MCE, HY-K0010) and 1mM phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich, 32998-6). Protein concentrations were measured using the Pierce^TM BCA Protein Assay Kit (Thermo fisher, 23227) and equal amounts of proteins were loaded on SDS-PAGE gels. Immunoblotting analysis was performed as described previously 32 (link). The following primary antibodies were used with indicated dilution: Kindlin-2 (Proteintech, 11453-1-AP, 1:1000), DDX3X (Proteintech, 11115-1-AP, 1:1000), c-Myc (Cell signaling, 5605S, 1:1000), GAPDH (ABclonal, AC035, 1:5000), GFP (Transgen, HT801, 1:2000), Flag (Sigma, F1804, 1:3000), GST (Transgen, HT601, 1:2000) and MBP (Cell signaling, 2396S, 1:1000).

Primary antibodies used were against: Caprin1 (Proteintech 15112-1-AP, 1:500), HuR (Santa Cruz Biotechnology SC-5261, 1:500), G3BP1 (Santa Cruz Biotechnology SC-365338, 1:500), G3BP1 (Abcam ab556574, 1:500), Dcp1a (Abcam ab57654, 1:500), eIF3η (Santa Cruz Biotechnology SC-137214, 1:100), eIF4G (Cell Signaling Technology 2498S, 1:100), DDX3X (Proteintech 11115-1-AP, 1:100), RACK1 (BD Transduction Laboratories 610177, 1:100), GAPDH (Proteintech 60004-1-Ig, 1:50,000) T-eIF2α (Cell Signaling Technology 9722S, 1:1000) and eIF2α [pSer52] (Enzo BML-SA405-0100, 1:1000).
Second antibodies used were: goat-anti-rabbit IgG conjugated to Alexa Fluor 633 (Invitrogen A21070, 1:500), goat-anti-mouse IgG conjugated to Atto-550 (Sigma, 1:1000), goat-anti-mouse IgG2a conjugated to Alexa Fluor 488 (Invitrogen A21131, 1:1000), goat-anti-mouse IgM conjugated to Alexa Fluor 633 (Invitrogen A21046, 1:500), goat-anti-rabbit IgG conjugated to Alexa Fluor 546 (Invitrogen A11035, 1:1000), DAPI (chromatin labelling, 1 mM 1:1000), donkey-anti-mouse-HRP (Jackson ImmunoResearch 715-035-150, 1:10,000), donkey-anti-rabbit-HRP (Jackson ImmunoResearch 711-035-152, 1:10,000).