Fig. 4

GKN2 directly interacts with Hsc70. a Hsc70 was identified by MS as interactor of GKN2. b GKN2 interacted with Hsc70 in GC cell lines. MGC cells expressing myc-GKN2 and SGC cells expressing myc-Hsc70 were lysed, subjected to immunoprecipitation using anti-myc antibody, and detected using indicated antibodies. Input: cell lysate without immunoprecipitation. c Confocal laser-scanning detected the expression of GKN2 and Hsc70 in GC cell lines. Scale bar: 50 μm. d Effect of Hsc70 knockdown. Cells were transfected with siRNA to knockdown Hsc70 and cultured for 48 h. Then indicated proteins were detected. e The stability assays of Hsc70 after overexpression of GKN2. The cells were treated with 10 μM cycloheximide (CHX) for 12 h then treated with H₂O₂ (300 uM, 6 h) before harvest. f The stability assays of Hsc70 after overexpression of GKN2 or its mutation. 293 T cells were transient co-transfected with Hsc70 and GKN2/GKN2 mutation/control plasmid and cultured for 24 h. Then cells were treated with 10 μM cycloheximide for the indicated times then treated with H₂O₂ (300 uM, 6 h) before harvest. g The expression and ubiquitination of Hsc70 after overexpression of GKN2. Cells were transient transfected with GKN2 and/or HA labeled ubiquitin enzyme (Ub-HA) and cultured for 24 h. With exposure to MG132 (5uM, 2 h), cells were treated with H₂O₂ (300 uM, 6 h) before harvest. Then cells were lysed, subjected to immunoprecipitation using anti-Hsc70 antibody and detected using indicated antibodies. MG132: proteasome inhibitor