Fig 2: Effect of miR-23b-3p on the activation of Gab1 and Fak. (a) Gab1, Fak, p-Gab1(Tyr307), and p-Fak (Tyr397) levels in HaCaT, C33A and CaSki cells. (b) The overexpression of miR-23b-3p significantly reduces the activation of Gab1 and Fak in CaSki cells. The expression of the proteins was normalized with the GAPDH load control. N/T: non-transfected cells. The data are expressed as averages ± SE. *p < 0.05.

Fig 3: The N-terminus of Gab1ß directs palmitoylation and is required for membrane localisation and function. (A) Schematic outlining the structure of the Gab1 PH domain and comparison of the N-terminus of Gab1ß with corresponding regions of other Gab-related proteins. The arrangement of the seven ß-sheets and the a-helix, and relative positions of a putative nuclear localisation sequence (NLS) and PIP3 binding residues are shown. Cysteines within the a-helix are highlighted in bold. (B) EGFP immunoprecipitates from ESCs stably transfected with EGFP or Gab1-EGFP fusion proteins cultured overnight with [3H]palmitate, were fractionated on an SDS protein gel, transferred to a filter and autoradiographed (top) and probed for EGFP protein (bottom). CA3 designates variants in which the three N-terminal cysteines of Gab1ß are substituted with alanine. (C) Confocal images of EGFP fluorescence in ESCs transfected with the 47-EGFP, 47CA3-EGFP, PH-domain EGPF and PH-domain CA3-EGPF fusion proteins (counterstained with DAPI). Scale bars: 10 µm. (D) Growth curves of Gab1ß knockout (KO) ESCs and Gab1ß KO ESCs stably transfected with either Gab1ß wild-type or CA3 mutant cDNA expression vectors. The graphs represent the means±s.e.m. obtained from three independent experiments. Student's t-test analysis showed significant differences between the KO and Gab1ß-expressing lines from day 4 to day 6 (P<0.0001), but not between KO and KO-Gab1ß:CA3m cells.

Fig 4: Localisation of Gab1ß at the cell membrane. Confocal images of (A) wild-type ESCs, (B) Gab1ß knockout ESCs and (C) Gab1ß knockout ESCs stably transfected with a Gab1ß cDNA expression vector, immunostained with antibodies against Gab1 (red) and counterstained for DNA with DAPI (blue). (D-I) Confocal images of EGFP fluorescence in ESC stably expressing the following Gab1-EGFP fusion proteins: (D) Gab1ß-EGFP; (E) Gab1a-EGFP; (F) N-terminal EGFP-Gab1ß; (G) Gab1ß-EGFP fusion lacking 15 N-terminal amino acids; (H) the 47 N-terminal amino acid peptide of Gab1ß fused to EGFP; and (I) 15 N-terminal amino-acid peptide of Gab1ß fused to EGFP. White arrows highlight consistently observed intracellular accumulations of Gab1ß proteins. Scale bars: 10 µm.

Fig 5: Gab1ß as an ESC signal transducer. (A) Western blot of Gab1 immunoprecipitates from unstimulated ESCs (-), and ESCs stimulated with serum (sr) or LIF (lf) for 10 min, probed with Gab1pY627, Gab1pY307, phosphotyrosine and Gab1 specific antibodies. The dashes indicate position of 100 kDa MW marker. (B) Western blot of SHP2 immunoprecipitates from cells treated as in A probed with antibodies to Gab1 and SHP2. (C) Western blot of cell lysates from wild-type ESCs, and Gab1ß knockout ESC line stably transfected with empty vector (-/-), Gab1ß, and Gab1a expression vectors, unstimulated or following stimulation with LIF for 15 min. The blot was incubated with antibodies to phospho-Akt, Akt, phospho-Erk, Erk1/2, phospho-Stat3 and Gab1.