Fig 1: Microtubules are involved in TBC1D3-mediated regulation of EGFR ubiquitination.(A) SMMC7721 cells were seeded in four 60-mm dishes. The following day, cells were transfected with control HA vector (dishes 1 and 2), HA-TBC1D3 (dish 3) and HA-TBC1D3(?320–353) (dish 4), respectively. The next day cells were serum-starved and then treated with 100 ng/ml of EGF for 15 minutes. Lysates were immunoprecipitated (IP) with anti-EGFR antibody (dishes 2, 3 and 4) or control IgG (dish 1). After SDS PAGE, the blots were stained with Ponceau S (bottom panels) followied by immunoblotting (IB) with anti-c-Cbl, anti-EGFR, anti-HA and anti-ubiquitin antibodies (top panels). Anti-ubiquitin and anti-HA antibodies were used to monitor ubiquitinated EGFR, HA-TBC1D3 and its mutant. Molecular weight markers are shown on left in kDa. WCL, whole cell lysate. (B) The bar graph is derived from densitometric analysis of Western blots as typified in top right panel in A. Ubiqitinated EGFR and c-Cbl are normalized to EGFR total amounts. The data are presented as means ± SD of three independent experiments (**, p<0.01).