Rabbit anti ha

HepG2 cells (a gift from Pingsheng Liu’s laboratory), COS-7 cells, U2OS cells, and HEK293T cells were maintained in DMEM (Invitrogen) supplemented with 10% FBS at 37°C in 5% CO₂. Transfections were performed using Lipofectamine 3000 (Invitrogen) for DNA plasmids, except for transfection of plasmids after delipidation, which was performed using X-tremeGENE HP DNA Transfection Reagent (Roche) and Lipofectamine RNAi MAX (Invitrogen) for siRNAs according to the manufacturer’s instructions. Cell samples were harvested 24 h after plasmid transfection or 48 h after siRNA transfection unless otherwise indicated. Immunofluorescence experiments were performed as described previously. Transfected cells were grown on coverslips and immunostained with primary antibodies and various Alexa Fluor–conjugated secondary antibodies (Invitrogen). Images were captured at RT on a 3D–structured illumination microscopy (3D-SIM) or Zeiss LSM700 confocal microscope as indicated. The primary antibodies used are mouse–anti-HA (ORIGENE; TA180128), rabbit–anti-HA (Abcam), rabbit–anti-Myc (Sigma; C3956), mouse–anti-Flag (Sigma), rabbit–anti-calreticulin (Abcam; ab2907), mouse–anti–Climp-63 (Enzo; ENZ-ABS669-0100), rabbit–anti–septin 2 (Abcam; ab187654), and mouse–anti–α-tubulin (Abcam; ab7291).

HEK293T cells were transfected with Lipofectamine 2000 (Invitrogen) for both co-immunoprecipitation and luciferase assay. Co-IP and immunoblot were performed as previously described 50 (link). The antibodies used include mouse anti-HA (Covance), rabbit anti-HA (Abcam), mouse anti-FLAG (Sigma), and rabbit-anti-GFP (Cell signaling). For luciferase assay, each transfection was triplicated with 400 ng wild type or mutant Gen1, 100 ng pCX-EGFP, 200 ng HA-Six1, 200 ng HA-Eya1, and 200 ng Gdnf-luc2. Luciferase activity was scored 48 hours after transfection and calibrated by the percentage of GFP positive cells (determined by FACS). Each experiment was performed at least twice independently.