pegfr (tyr1068) antibody  (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

Journal: Cancer Management and Research

Article Title: ArfGAP with the SH3 Domain, Ankyrin Repeat and PH Domain 1 Inversely Regulates Programmed Death-Ligand 1 Through Negative Feedback of Phosphorylated Epithelial Growth Factor Receptor and Activation of Nuclear Factor-Kappa B in Non-Small Cell Lung Cancer

doi: 10.2147/CMAR.S493368

Figure Lengend Snippet: Changes in the expression of activated receptor tyrosine kinases (RTKs) when AMAP1 was knocked down: The investigation of changes in activated RTK expression when AMAP1 was knocked down in HCC4006 ( A ) and A549 ( B ) cells using the activated RTKs assay. Each antibody was plotted in pairs, with the first six and last two representing the positive control. For A549 cells, owing to the low PD-L1 expression, stimulation was performed with TGF-β 1 ng/mL. Western blot analyses of pEGFR, pSyk, and pACK1 in HCC4006 ( C ) and A549 ( E ) cells. A comparison of pEGFR expression in HCC4006 ( G ) and A549 ( I ) cells when AMAP1 was knocked down, as observed through IF. Each result was semi-quantified using the ImageJ software ( D and F ) and the analysis application of BZ-X810 ( H and J ). The vertical axis represents the band ratio normalized to the brightness of the negative control (set to 1) ( D and F ). The vertical axis of the graph represents the cell count, and the brightness per cell was measured ( H and J ).

Article Snippet: Antigen recognition was performed by incubating the cells with primary antibodies against pEGFR (Cell Signaling Technology; cat. no. 3777 1:1000), PD-L1 (Cell Signaling Technology; cat. no. 13684 1:1000) and DDEF1 (Santa cruz; cat. sc-374410 1:1000) for 20 hours at 4°C, followed by incubation with Alexa Fluor 568‐ (Invitrogen; Thermo Fisher Scientific, Inc. A-10037,1:1000) and 488‐ (Invitrogen; Thermo Fisher Scientific, Inc. A‐21441 1:1000) conjugated secondary antibodies.

Techniques: Expressing, Positive Control, Western Blot, Comparison, Software, Negative Control, Cell Counting

Journal: Cancer Management and Research

Article Title: ArfGAP with the SH3 Domain, Ankyrin Repeat and PH Domain 1 Inversely Regulates Programmed Death-Ligand 1 Through Negative Feedback of Phosphorylated Epithelial Growth Factor Receptor and Activation of Nuclear Factor-Kappa B in Non-Small Cell Lung Cancer

doi: 10.2147/CMAR.S493368

Figure Lengend Snippet: Results of inhibiting pEGFR using the EGFR inhibitor osimertinib: Western blot analysis showing the inhibition of pEGFR in HCC4006 ( A ) cells using osimertinib. The concentration of osimertinib was set at 25 nM. Western blot analysis demonstrating the inhibition of pEGFR in A549 ( C ) cells using osimertinib. The concentration of osimertinib was set at 2 μM. For A549 cells, owing to the low PD-L1 expression, stimulation was performed with TGF-β 1 ng/mL. Each result was semi-quantified using the ImageJ software ( B and D ). The vertical axis of the graph represents the band ratio normalized to the brightness of the negative control (set to 1).

Article Snippet: Antigen recognition was performed by incubating the cells with primary antibodies against pEGFR (Cell Signaling Technology; cat. no. 3777 1:1000), PD-L1 (Cell Signaling Technology; cat. no. 13684 1:1000) and DDEF1 (Santa cruz; cat. sc-374410 1:1000) for 20 hours at 4°C, followed by incubation with Alexa Fluor 568‐ (Invitrogen; Thermo Fisher Scientific, Inc. A-10037,1:1000) and 488‐ (Invitrogen; Thermo Fisher Scientific, Inc. A‐21441 1:1000) conjugated secondary antibodies.

Techniques: Western Blot, Inhibition, Concentration Assay, Expressing, Software, Negative Control