Anti flag dykddddk

After fixation in 4% paraformaldehyde/PBS and permeabilization, cells were incubated with antibodies diluted in PBS containing 1% BSA (Nacalai) and 0.02% Tween‐20 for 1 h. Nuclear staining was performed using DAPI (Dojindo). Images of stained cells were acquired with a laser confocal microscope (Carl Zeiss LSM710 and LSM800 or Olympus FV3000). In case of CXCL12 treatment, cells were cultured in DMEM supplemented with 0.05% BSA and treated with 100 ng/ml CXCL12. The proximity ligation assay (PLA) of transfected Cos7 cells was done using anti‐Halo rabbit antibody (Promega) and anti‐FLAG M2 mouse antibody (Sigma‐Aldrich) along with Duolink PLA kit (Sigma‐Aldrich) according to the manufacture’s protocol. The used antibodies were as follows: anti‐X123 (3C7), anti‐Epsin1 (C‐11), anti‐CXCR4 (12G5), and anti‐GAPDH (0411) antibodies (Santa‐Cruz Biotechnology); anti‐Clathrin (D3C6), anti‐Rab5 (E6N8S), anti‐Rab7 (D95F2), anti‐Rab11 (D4F5), anti‐ITCH (D8Q6D), anti‐HA tag, anti‐FLAG (DYKDDDDK), and anti‐Myc tag antibodies (Cell Signaling Technology); anti‐CXCR4 antibody (Abcam); anti‐ubiquitin (FK2) antibody (MBL); specific secondary antibodies conjugated to Alexa Fluor 350, 488, 555 or Alexa Fluor Plus 647 (Invitrogen).

Total cell extracts were prepared by dissolving cells with 1×Laemmli SDS reducing buffer (50 mM Tris-HCL [pH 6.8], 2% SDS and 10% glycerol) and boiled for denaturation. Equal amounts of protein sample were separated on 4-15% Mini-PROTEAN TGX^TM Precast Gel (Bio-Rad, Hercules, CA, USA). Anti-Cyclin A2 (#4656S, Cell Signaling, Danvers, MA, USA), anti-β-actin (#sc-47778, Santa Cruz Biotechnology, Dallas, TX, USA), anti-KRAS (#sc-30, Santa Cruz Biotechnology), anti-cleaved PARP (#9541S, Cell Signaling Technology), anti-phospho-JNK (#4668S, Cell Signaling Technology), anti-cleaved Caspase-3 (#9661S, Cell Signaling Technology), anti-PLK1 (#4513S, Cell Signaling Technology), anti-FLAG (DYKDDDDK)(#2368S, Cell Signaling Technology) and anti-GAPDH (#60004-1-Ig, Proteintech Group, Rosemont, IL) antibodies were used as primary antibodies. Peroxidase-AffiniPure Goat Anti-Rabbit IgG (#111-035-144) and Anti-Mouse IgG (#115–035–003, Jackson ImmunoResearch, West Grove, RA, USA) were used as secondary antibodies. Antibody binding was visualized by SuperSignal West Pico/FemtoChemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and X-ray films (AGFA-Gevaert, Mortsel, Belgium).

The following antibodies were used: anti‐DYKDDDDK/flag (filter trap 1:1,000, FACS 1:250, Cell Signaling), anti‐myc (1:1,000, clone 9E10, Santa Cruz), anti‐HA (1:1,000, clone 3F10, Roche), anti‐GFP (1:1,000, clone N86/8, NeuroMab), anti‐GA clone 5F2 (1 μg/ml) (Mackenzie et al, 2013), mouse IgG2a (1 μg/ml, Sigma), and rabbit IgG (1:250, Sigma).