Additional file 4 of The phosphatase PPM1F, a negative regulator of integrin activity, is essential for embryonic development and controls tumor cell invasion

Additional file 4: Figure S4 shows representative western bots of integrin β1 immunoprecipitations and the corresponding four repetitions of the analysis of integrin β1 phosphorlyation in integrin β1 immunoprecipitates generated from PPM1F WT and PPM1F KO cells. To illustrate surface exposed integrins, cells were biotinylated and streptactin pulldown were performed. Furthermore, integrin β1 immunoprecipitates from PPM1F KO cells were treated with EndoH to analyse N-glycan composition. Figure S4: Related to Fig. 7. (A) Integrin β1 was immunoprecipitated from lysates of A172 wildtype and PPM1F KO cells using monoclonal antibody P5D2. As a control, immunoprecipitation was performed with an isotype-matched control antibody (IgG). Lysates and IPs were first probed with rabbit-monoclonal anti integrin β1 antibody D2E5. (B) Lysates as in (A) were used to immunoprecipitate integrin β1 with the mouse monoclonal antibody P5D2 (left lanes) or the rat monoclonal antibodies AIIB2 and 9EG7 (right lanes). Western Blot was performed with the polyclonal phospho-specific antibody against pT788/pT789 integrin β1 (upper panel) and upon stripping of the membrane against integrin β1 (lower panel). The bar graph depicts the densitometric evaluation pT788/pT789 signals vs. total integrin β1 observed in five repetitions of this experiment. (C) A172 wildtype and PPM1F KO cells (clone 1) were surface biotinylated and following lysis, biotinylated proteins were collected by streptavidin beads. The lysates containing biotinylated proteins and the streptavidin pull-downs (strep.) were probed as in (B) with antibodies against pT788/pT789 integrin β1 (upper panel) and against integrin β1 (lower panel). (D) Integrin β1 was immunoprecipitated from lysates of A172 wildtype and PPM1F KO cells using monoclonal antibody P5D2. As a control, immunoprecipitation was performed with an isotype-matched control antibody (IgG). Lysates and IPs were first probed with the polyclonal phospho-specific antibody against pT788/pT789 integrin β1 (upper panel) before stripping and reprobing with rabbit-monoclonal anti integrin β1 antibody D2E5 (lower panel). Shown are four repetitions of the experiment depicted also in (B). (E) Integrin β1 immunoprecipitates from lysates of PPM1F KO cells as in (A) were treated in vitro with Endoglycosidase H (EndoH) for 1 h before Western Blotting with rabbit-monoclonal anti integrin β1 antibody D2E5. Arrowheads point to the slow migrating integrin β1 band not modified by EndoH, and the faster migrating band, which has a lowered apparent molecular weight and increased mobility upon EndoH treatment.