Additional file 2 of The phosphatase PPM1F, a negative regulator of integrin activity, is essential for embryonic development and controls tumor cell invasion

Additional file 2: Figure S2 Provides evidence that fibroblasts isolated from ppm1f-/- mice do not exhibit altered integrin surface levels or focal adhesion protein expression and details the quality control of the polyclonal anti-mousePPM1F antiserum and the polyclonal anti pT788/pT789 integrin β1 antiserum. Figure S2: Related to Fig. 4. Fibroblasts isolated from ppm1f-/- mice do not show altered focal adhesion protein expression or increased integrin surface levels. (A) WCLs of indicated MEF cell lines were analyzed by Western blotting using the purified “in-house” generated anti-mouse PPM1F antibody. As controls, WCLs of 293 T cells transfected with the empty vector (mock), GFP (GFP) or murine PPM1F (mWT) were loaded. The antibody specifically recognizes the ~ 60 kDa mouse PPM1F, but also cross-reacts with the ~ 55 kDa human PPM1F with lower specificity. Human PPM1F is endogenously expressed in HEK cells (first three samples) and re-expressed in its wildtype and phosphatase-dead form in the MEF PPM1F -/- cells (last two samples). In addition to PPM1F, a non-specific band at ~ 70 kDa is detected (*). (B) Lysates from mouse embryonic fibroblasts isolated from PPM1F + / + or littermate PPM1F-/- embryos at E10.5. PPM1F-/- cells were stably transduced with wildtype human PPM1F (hWT) or human PPM1F D360A (hDA). WCLs were subjected to Western blotting with the indicated antibodies against a panel of focal adhesion proteins; monoclonal α-tubulin antibody was used as loading control. (C) MEF cell lines from (B) were analyzed by flow cytometry for integrin surface expression levels. Cells were stained with the indicated integrin-specific antibodies. Unstained wildtype MEFs or MEFs stained with an isotype-matched irrelevant antibody (IgG control) served as controls; count ≥ 10 000 cells. (D) MEF cells were stained for the indicated integrin subunits with integrin-specific antibodies and analyzed by flow cytometry for surface expression levels; count ≥ 10 000 cells. Mean fluorescence intensity derived from one representative experiment is shown (MFI). (E) 2 µg GST (control) or GST-integrin β1 cytoplasmic domain in its wildtype, single-T788D or double-T788D/T789D mutated form were incubated with or without recombinant CaMKIIβ (120 ng) in the presence of ATP/calmodulin/CaCl2 at 30 °C for 60 min. Reactions were stopped via addition of SDS sample buffer and subjected to Western blotting using the indicated antibodies and Coomassie staining.