Figure 2

β4 phosphorylation occurs abundantly on the β4 CS. (A) Representation of the tyrosines (Y) present and mutated to phenylalanines (F) in the connecting segment (CS) of the various β4 mutants (4Y-F, 2Y14F and 2Y13F) and wild type (WT) β4. (B) EGF-induced β4 tyrosine phosphorylation of β4 tyrosine mutants shown by Western blot analyses of β4 immunoprecipitation (IP) samples of EGF-treated and untreated COS7 cell overexpressing WT or mutant β4 together with the EGFR. Blots were probed for β4 and phospho-tyrosine (pY). (C) Comparable β4 surface levels in A431 cell lines analyzed by FACS. The A431 cell lines analyzed are as follows: WT, expressing endogenous β4; KO, depleted of endogenous β4 by CRISPR-Cas9; Resc, β4 (KO) cells reconstituted with WT β4; 4Y-F, 2Y13F and 2Y14F β4 mutants, β4 (KO) cells reconstituted with mutated β4. (D) Representative Western blot analyses of WCLs of the various A431 cells for phosphorylation of β4 at Y1440 and Y1422 using phosphosite-specific antibodies. Cells were untreated or treated with EGF for 5 min after 20 h serum starvation. (E) Double immunofluorescence staining for β4 and plectin, and β4 and the EGFR in A431 β4 (KO) cells and β4 (KO) cells reconstituted with WT β4, 4Y-F or 2YF14 β4. Scale bars: 10 µm. (F) Representative Western blot analyses of β4 tyrosine phosphorylation and total β4 levels in β4 IP samples obtained from the various A431 cells untreated or treated with EGF for 5 min after 20 h serum starvation. (G) Quantification of β4 tyrosine phosphorylation levels in the various A431 cell lines, per sample normalized to total β4 levels and per experiment to the pY/β4 WT levels. Graphs shows the mean + SD from 3 independent experiments. Uncropped images of western blots, and merged chemiluminescent and colorimetric blots obtained with a ChemiDoc imaging system (BioRad) are shown in Suppl. Fig. 2.