anti-pmek (41g9)  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: E-cadherin mechanotransduction activates EGFR-ERK signaling in epithelial cells by inducing ADAM-mediated ligand shedding

doi: 10.1101/2025.03.06.641828

Figure Lengend Snippet: (A) Schematic representation of the EGFR-ERK signaling pathway. Ligand binding to the EGFR extracellular domain induces the sequential activation of EGFR, Ras, RAF, MEK and ERK. (B and C) Western blot of lysates from non-strained and strained (15 minutes) MDCK cells, probed for MEK1 and MEK2 phosphorylated on Ser 217 and Ser 221 [pMEK1/2 (Ser 217 /Ser 221 )], ERK1 and ERK2 phosphorylated on Thr 202 and Tyr 204 [pERK1/2 (Thr 202 /Tyr 204 )], total ERK1/2 and α-Tubulin. Quantifications show ratios of pERK/α-Tubulin (n = 6 independent experiments), pMEK/α-Tubulin (n = 7 independent experiments) and pEGFR/loading control (either p130Cas or α-Tubulin) (n = 13 independent experiments), normalized to the mean ratio of the non-strained control. Normalization of pERK to total ERK and pMEK to total MEK shows comparable induction of pERK and pMEK by strain, respectively (see fig. S2Aand S2B). (D and E) Western blot of lysates from non-strained and strained (15 minutes) MDCK cells, in the presence or absence of Afatinib (1 μM), and probed for pERK1/2 (Thr 202 /Tyr 204 ), α-Tubulin, pEGFR (Tyr 1068 ) and p130Cas as indicated.Graphs show the ratio of pERK/α-Tubulin (n = 6 independent experiments) and pEGFR/p130Cas (n = 5 independent experiments), normalized to the mean ratio of the non-strained, non-treated control. (F) Western blot of lysates from non-strained and strained (15 minutes) DLD1 cells, in the presence or absence of Cetuximab (5 μg/mL) or Panitumumab (20 μg/mL), and probed for pERK1/2 (Thr 202 /Tyr 204 ) and α-Tubulin. The quantification shows the ratio of pERK/α-Tubulin, normalized to the mean ratio of the non-strained, non-treated control (n = 5 independent experiments). (G) Western blot of lysates from MDCK, MDCK-T151-E-cadherin and MDCK-ΔVBS-α-catenin cells, either strained (15 minutes) or unstrained, and probed for pERK1/2 (Thr 202 /Tyr 204 ) and α-Tubulin. The quantification shows the ratio of pERK/α-Tubulin in WT MDCK (n = 7 independent experiments), MDCK-T151-E-cadherin (n = 7 independent experiments) and MDCK-ΔVBS-α-catenin cells (n = 6 independent experiments), normalized to the mean ratio of the non-strained, WT MDCK control. (H) Western blot of lysates from MDCK and MDCK-T151-E-cadherin cells, either strained (15 minutes) or unstrained, and probed for pEGFR (Tyr 1068 ) and p130Cas. The quantification shows the ratio of pEGFR/p130Cas, normalized to the mean ratio of the non-strained, WT MDCK control (n = 6 independent experiments). All statistical tests shown are paired ratiometric t-tests. * p < 0.05, ** p < 0.01, *** p < 0.005.

Article Snippet: Proteins of interest were probed by overnight incubation with primary antibodies at 4°C. pERK (Cell Signaling Technology, 4370), ERK (Cell Signaling Technology, 4370), and pMEK (Cell Signaling Technology, 9121) antibodies were used 1/2500. pEGFR (Cell Signaling Technology, 2234), EGFR (Cell Signaling Technology, 4267) and MEK (Cell Signaling Technology, 9122) antibodies were used 1/1000. α-Tubulin (Sigma, T9026) and p130Cas (BD Biosciences, 610272) antibodies were used 1/5000.

Techniques: Ligand Binding Assay, Activation Assay, Western Blot, Control