Fig 2: Candidalysin-induced adaptor activation requires EGFR and MMP activity. Inhibition of EGFR activity suppresses (A) C. albicans- and (B) candidalysin (Clys)-induced adaptor activation. Use of Gefitinib or PD153035 EGFR TK inhibitors suppresses WT C. albicans–induced and Clys-induced activation of Gab1, Grb2, Shc, Shp2, and c-Cbl. C, MMP inhibition suppresses candidalysin-induced adaptor activity. Pretreatment of TR146 cells with Marimastat (10 or 1 μM, designated M10 or M1) suppressed Clys-induced phosphorylation of Gab1, Grb2, Shc, Shp2, and c-Cbl. Protein lysates were isolated 2 h post stimulation. Blots are representative of two (Grb2) or three (Gab1, Shc, Shp2, and c-Cbl) independent experiments. (Note: because data from other experiments presented in this article were originally run on the same gel, the α-actin loading control is reused in Fig. 2, A–C). MMP, matrix metalloproteinase.

Fig 3: Western blot analysis of PLSCR1, Shc, Src, and cyclin D1 expression in colorectal tissue pairs. Twenty-two tissue pairs were utilized to evaluate protein expression. The expression of each protein in tumor (T) versus normal (N) tissues was evaluated with the nonparametric Mann–Whitney test (p < 0.05). The raw data points are presented as a scatter plot, and each mean value is indicated with a horizontal line.

Fig 4: EGFR ligand binding is required for C. albicans and candidalysin-induced adaptor activation. Pretreatment of TR146 cells with the anti-EGFR mAb, Cetuximab suppressed phosphorylation of Gab1, Grb2, Shc, Shp2, and c-Cbl following (A) C. albicans infection or (B) candidalysin (Clys) stimulation. Blots are representative of two (Grb2) or three (Gab1, Shc, Shp2, and c-Cbl) independent experiments. (Note: because data from other experiments presented in this article were originally run on the same gel, the α-actin loading control is reused from Fig. 3, A and B).

Fig 5: Expression of LTK constructs in 293T cells.(A) Relative protein expression of wildtype and mutant LTK constructs in 293T cells. Lysates of 293T cells transiently expressing either HA-tagged vector control (v, lane 1), wildtype LTK (wt, lane 2), LTK-R669Q (RQ, lane 3), or LTK-F568L (FL, lane 4) were immunoblotted with an anti-HA antibody as well as HSP90 as a loading control. (B) 293T cell lysates containing the LTK proteins were treated with PNGase F (lanes 3, 5, and 7) or left untreated (lanes 2, 4, and 6) and immunoblotted with HA antibodies and HSP90 as a loading control. (C) HA-tagged LTK proteins expressed in 293T cells were immunoprecipitated and immunoblotted with HA antibodies as well as anti-phosphotyrosine (pTyr) antibodies (top). Antibodies that recognize the indicated proteins (Shc, pShC-Y239/Y240, ERK, pERK-T202/Y204, JAK1, pJAK1-Y1022/Y1023, JAK2, pJAK2-Y1007/Y1008, AKT, and pAKT-S473) were used to immunoblot cell lysates of 293T cells expressing LTK proteins (bottom). The phosphorylated form of the protein is designated with a “p” preceding the protein name.