Fig 2: 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 3: Laminin a2ß1?1 (Lm211) inhibits neuregulin 1 type III (Nrg1III)-induced ErbB2/3 and Grb2-Associated Binder-1 (Gab1) activation by negatively regulating protein kinase A (PKA).(A) Schematic representation of the hypothesis that Lm211 inhibits ErbB2/3 and Gab1 downstream of Nrg1III by negatively regulating PKA. (B) The PKA inhibitor H89 dose-dependently suppressed dbcAMP-induced ErbB2 and Gab1 phosphorylation in rat Schwann cells (SCs). dbcAMP was used for 3 days (n = 3, *p < 0.05; **p < 0.001 by Student t test). (C) Intermuscular injection of HB9 and KT5720 for 4 days reduced PKA activity, revealed by a decrease in PKA phospho-substrates. (D, E) Similar treatment significantly reduced ErbB2 and Gab1 activation in Nrg1IIItg//Lama2-/- and, to a lesser extent, in the other genotypes. The experiments were repeated at least 5 (D) or 3 (E) animals per genotype. *p < 0.05, ***p < 0.005, ***p < 0.001 by Student t test. The numerical data used in B, D-E, are included in S1 Data.

Fig 4: Expression profile of a novel Gab1 variant protein. (A) Gab1 western blot analysis of whole cell lysates from ESCs (ES), embryonal carcinoma (EC), 10T1/2 embryonic fibroblasts (fb) and pooled E12.5 embryo tissues (em). The positions of the 110 and 95 kDa Gab1 proteins are indicated. (B) Schematic of the Gab1 gene locus, showing the location of the transcription start sites for Gab1a and Gab1ß, and the exons. (C) Schematic showing the structure of Gab1a and Gab1ß proteins. Dark grey and light grey areas are proline rich-regions and the Met binding domain (MBD), respectively. (D) Gab1 and Oct4 western blots of whole cell lysates of ESCs (ES), primary embryonic fibroblasts (fb), pre-iPS cells (pr), iPSCs reprogrammed in 2i medium (2i) or serum/LIF medium (sr) and an epiblast stem cell line (Epi). (E) Quantitative RT-PCR analysis of Gab1ß and Gab1a expression in ESCs (ES) transitioned into epiblasts stem cells (Epi) and embryoid bodies (EB). Results represent means±s.d. from one experiment. Expression is normalised relative to the level in ESCs.

Fig 5: 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.