Anti gapdh 0411

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).

Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco’s Modified Eagles Medium (DMEM, Hyclone) supplemented with 1% penicillin/streptomycin (P/S) and 10% fetal bovine serum (Hyclone). Predesigned siRNA duplexes were purchased from Bioneer, and 10 nM siRNA transfection was carried out using RNAiMAX (Invitrogen, Waltham, MA, USA). Cells and tissues were lysed with N-PER™ and T-PER™ lysis buffer (Thermo Scientific, Waltham, MA, USA) supplemented with protease inhibitor cocktail (Roche, Hoffmann-La Roche AG, Basel, Switzerland), respectively. Lysates were resolved by SDS-PAGE gels and transferred onto nitrocellulose (NC) membranes (Merck Millipore, Burlington, MA, USA), and then antibodies were detected using the D-Plus ECL Femto System (Dongin Bio, Seoul, Korea) and the ImageQuant LAS4000 (GE Healthcare, Chicago, IL, USA). Antibodies used for immunoblotting included the following: anti-Ubiquitin (P4D1, Santa Cruz, Santa Cruz Biotechnology, Dallas, TX, USA), anti-UBE2H (18-Z, Santa Cruz), anti-Parkin (PRK8, Santa Cruz), anti-GAPDH (0411, Santa Cruz), anti-β-actin (C4, Santa Cruz), anti-Tau (Abcam, Cambridge, UK), and anti-UBE2L6 (MyBioSource, San Diego, CA, USA).