Figure 6.

Epidermal growth factor receptor (EGFR)-mediated phosphorylation regulates ABCA1 ubiquitination and is enhanced by TXLNA deficiency during testicular phagocytosis. (a) Primary cultured SCs were incubated with apoptotic GCs for 45 min. Subsequently, cells were harvested and whole-cell lysates were immunoblotted with phospho-EGFR (Tyr845, Tyr1068, Tyr1173 and Ser1046/7) and EGFR. (b) SCs were incubated with apoptotic GCs for 45 min, followed by immunofluorescence analysis of colocalization of EGFR and ABCA1. Nuclear are demonstrated using DAPI staining. (c) Ctrl siRNA or Txlna siRNA-transfected SCs were incubated with apoptotic GCs, in the presence or absence of 10 μM EGFR inhibitor (PD168393), for 6 h. Cells were then harvested and subjected to immunoprecipitation of EGFR with ABCA1. Immunoblot analyses of expression levels of ABCA1, pEGFR, EGFR and tubulin were also performed in the total-cell lysates (TCL). (d) Immunoprecipitation of EGFR with anti-ABCA1 in HeLa cells cotransfected with His-ABCA1 and indicated EGFR plasmid. After O/N serum starvation, cells were treated ±EGF (50 ng/ml, 30 min). Cells transfected with EGFRΔ790M were either treated with DMSO (PD168393 '-') or (10 μM, 6 h; PD168393 '+'). (e) Immunoprecipitation of ABCA1 with different phospho-specific antibodies in SCs in conditions shown in (c). (f) Immunoprecipitation of ABCA1 with pABCA1^Ser2054 in SCs in conditions shown in (d). (g) Immunoprecipitation of ABCA1 with Ub in SCs transfected with His-ABCA1, HA-Ub as well as indicated EGFR plasmid in conditions shown in (d). (h) SCs transfected with Ctrl siRNA or Txlna siRNA were pretreated with an Ub E1 inhibitor (PYR-41, 50 μM) for 2 h, followed by analysis of the binding and phagocytic index as described in Materials and Methods. Values represent the mean±S.E.M. of three independent experiments. (i) SCs transfected with Ctrl siRNA or Txlna siRNA were pretreated with PD168393 inhibitor (10 μM) for 6 h, followed by the binding and phagocytic analysis as described in (h)