Journal: bioRxiv

Article Title: Flotillin-2 regulates EGFR activation, degradation, and cancer growth

doi: 10.1101/2022.03.11.483779

Figure Lengend Snippet: (A) SILAC MS scheme shows that HeLa cell proteins were labeled with stable isotopes to compare Cbl complexes without EGF (light, L) and with 30 min of 100 ng/mL EGF stimulation (heavy, H). Cbl was immunoprecipitated (IP) from differentially treated cell lysates and the IPs were mixed at 1:1 ratio. The mixed IP was either directly used to generate peptides for MS (1) or was separated by SDS-PAGE (2), divided into fractions as indicated and each fraction was analyzed separately with MS. (B) The heat maps show SILAC ratios comparing abundance of specific proteins in the complex with Cbl in EGF treated and control samples (H/L) in the descending order. The arrows indicate the candidates selected for further analysis with in-cell pEGFR ELISA. This data is from five independent experiments. (C) HeLa cells were transfected with control siRNA (siNeg) or siRNA targeting Cbl/Cbl-b (positive control), FLOT1 or FLOT2 and seeded on 96-well plate. Phospho-EGFR ELISA was used to detect the levels of EGFR phosphorylation at Y1173 upon 100 ng/mL EGF stimulation for indicated time periods. The pEGFR Y1173 signal was normalized to the Janus-green whole-cell staining signal and the average values for three experiments (± SEM) were plotted versus EGF stimulation time. (D) HeLa cells were transfected with control siRNA (siNeg) or siRNA targeting FLOT1, FLOT2 separately and together for 48 hours and then treated with 25 ng/mL EGF for the indicated time periods. The lysates were subjected to western blot analysis with the indicated antibodies. (E) EGFR phosphorylation upon EGF stimulation is shown as an average ratio of pEGFR/EGFR ± SEM calculated by densitometry analysis of western blot (as shown in D) for three independent experiments. (F) Degradation of EGFR upon EGF stimulation was calculated by densitometry analysis of western blot in D from seven independent experiments and plotted as an average ratio of EGFR/HSC70 ± SEM. EGFR levels in untreated samples (0 EGF) were set as 1. Asterisk (*) denotes p < 0.05, (**) indicates p < 0.01 as compared to the corresponding time points for siNeg using Student’s t -test. MW in kDa is shown to the left of the western blot panels.

Article Snippet: Upon treatment the cells were fixed in 4% formaldehyde and in-cell ELISA was performed according to the manufacturer’s protocol (kit 62205, ThermoFisher Scientific). pEGFR antibodies provided in the kit were replaced with pEGFR Y1173 antibodies (44-794G) from ThermoFisher Scientific.

Techniques: Labeling, Immunoprecipitation, SDS Page, Enzyme-linked Immunosorbent Assay, Transfection, Positive Control, Staining, Western Blot

Journal: Aging (Albany NY)

Article Title: Budding uninhibited by benzimidazoles-1 (BUB1) regulates EGFR signaling by reducing EGFR internalization

doi: 10.18632/aging.204820

Figure Lengend Snippet: BUB1 depletion stabilizes EGFR. ( A ) A549 cells were transfected with non-targeting control scrambled (NSS) siRNA or BUB1siRNA. 48 hours post-transfection cells were cross-linked with 100 μM DSS for 30 minutes followed by 30 ng/mL EGF for an additional 30 minutes. Resulting lysates were resolved on SDS-PAGE gels and probed with indicated antibodies. ( B ) Quantitation of western blots from ( A ) only the monomer species of EGFR is plotted. Control siRNA (NSS) transfected, EGF treated lanes were set as 1 fold and used as a baseline for estimating fold enrichment in other samples. ( C ) Quantitation of pEGFR and EGFR dimers from SDS and EGF treated lanes only (lanes 4 and 8 only in A ). NSS transfected lane was set as 1 fold and used as a baseline for estimating fold enrichment in BUB1 siRNA transfected samples. ( D ) Gene expression values from non-metastatic adenocarcinoma samples (N=331) from TCGA lung dataset were log 2 transformed and median centered and correlation coefficient (r) was calculated. BUB1 and EGFR expression is expressed as log 2 transformed values. Correlation coefficient and p-value are listed.

Article Snippet: Antibodies to pAKT (cat# 4060), total AKT (cat# 9272), pEGFR(Y845) (cat #2231), pEGFR(Y1092), pEGFR (Y1068) (cat# 3777), pEGFR (Y1173), total EGFR (cat # 4267; # 2232), Her2/ErbB2, c-Met, pFAK, FAK, pErk1/2 (42/44) (cat # 9101; cat # 4370), Erk1/2 (42/44) (cat # 4695) and GAPDH (all from Cell Signaling), β-adaptin (BD Biosciences, cat # 610382), EEA1 (Ab70521), and Actin were from Abcam.

Techniques: Transfection, SDS Page, Quantitation Assay, Western Blot, Expressing, Transformation Assay

Journal: Aging (Albany NY)

Article Title: Budding uninhibited by benzimidazoles-1 (BUB1) regulates EGFR signaling by reducing EGFR internalization

doi: 10.18632/aging.204820

Figure Lengend Snippet: BUB1 inhibition reduces EGFR activation. MDA-MB-231 ( A ) NCI-H358 ( B ) and MRC5 ( C ) cells were serum starved and pre-treated with 10 μM 2OH-BNPP1 for 1 hour followed by 50 ng/mL EGF. Cells were harvested at the indicated time-points (10-180 minutes) after EGF treatment. Whole cell lysates from these samples were resolved on SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% de-fatted milk-TBST and probed with pEGFR (Y845), pAKT (S473), and pERK1/2 antibodies. The blots were also probed with antibodies raised against total proteins.

Article Snippet: Antibodies to pAKT (cat# 4060), total AKT (cat# 9272), pEGFR(Y845) (cat #2231), pEGFR(Y1092), pEGFR (Y1068) (cat# 3777), pEGFR (Y1173), total EGFR (cat # 4267; # 2232), Her2/ErbB2, c-Met, pFAK, FAK, pErk1/2 (42/44) (cat # 9101; cat # 4370), Erk1/2 (42/44) (cat # 4695) and GAPDH (all from Cell Signaling), β-adaptin (BD Biosciences, cat # 610382), EEA1 (Ab70521), and Actin were from Abcam.

Techniques: Inhibition, Activation Assay, SDS Page

Journal: Aging (Albany NY)

Article Title: Budding uninhibited by benzimidazoles-1 (BUB1) regulates EGFR signaling by reducing EGFR internalization

doi: 10.18632/aging.204820

Figure Lengend Snippet: Inhibition of BUB1 kinase activity reduces EGFR active dimers without affecting inactive-EGFR dimers. A549 ( A ) MRC5 ( B ) and MDA-MB-231-1833 ( C ) cells were serum starved for 3-4 hours, pretreated with 2OH-BNPP1 (10 μM), erlotinib (10 μM) or cetuximab (50 μg/mL) for 1 hour followed by EGF (50 ng/mL) for 10 min. DSS (200 μM) was added for an additional 20 minutes. Total cell lysates were made, resolved on 3-8% gels and probed with pEGFR (Y845), EGFR, Her2 and c-Met antibodies.

Article Snippet: Antibodies to pAKT (cat# 4060), total AKT (cat# 9272), pEGFR(Y845) (cat #2231), pEGFR(Y1092), pEGFR (Y1068) (cat# 3777), pEGFR (Y1173), total EGFR (cat # 4267; # 2232), Her2/ErbB2, c-Met, pFAK, FAK, pErk1/2 (42/44) (cat # 9101; cat # 4370), Erk1/2 (42/44) (cat # 4695) and GAPDH (all from Cell Signaling), β-adaptin (BD Biosciences, cat # 610382), EEA1 (Ab70521), and Actin were from Abcam.

Techniques: Inhibition, Activity Assay

Journal: Aging (Albany NY)

Article Title: Budding uninhibited by benzimidazoles-1 (BUB1) regulates EGFR signaling by reducing EGFR internalization

doi: 10.18632/aging.204820

Figure Lengend Snippet: BUB1 inhibitor reduces endocytosis of active EGFR in MDA-MB-231-1833 cells. ( A ) Cells were plated on glass coverslips, serum starved for 3-4 hours and pretreated with 2OH-BNPP1 (10 μM) or erlotinib (10 μM) for 1 hour followed by EGF treatment (50 ng/mL). Cells were fixed at different time points (5, 20, 40 and 80 minutes) post EGF treatment and processed for staining with pEGFR (Y1068) and EEA1 antibodies. Representative confocal images 20 minutes post EGF treatment are shown. ( B ) co-localization of pEGFR (Y1068) with EEA1 was estimated on ImageJ using JACOMP plugin. Data at 20 min post EGF treatment is plotted. Two-sided students t-test was performed on MS-Excel (p values, ** = 0.00097, ***=3.17 X 10 -5 ).

Article Snippet: Antibodies to pAKT (cat# 4060), total AKT (cat# 9272), pEGFR(Y845) (cat #2231), pEGFR(Y1092), pEGFR (Y1068) (cat# 3777), pEGFR (Y1173), total EGFR (cat # 4267; # 2232), Her2/ErbB2, c-Met, pFAK, FAK, pErk1/2 (42/44) (cat # 9101; cat # 4370), Erk1/2 (42/44) (cat # 4695) and GAPDH (all from Cell Signaling), β-adaptin (BD Biosciences, cat # 610382), EEA1 (Ab70521), and Actin were from Abcam.

Techniques: Staining

Journal: JCI Insight

Article Title: Mevastatin promotes healing by targeting caveolin-1 to restore EGFR signaling

doi: 10.1172/jci.insight.129320

Figure Lengend Snippet: (A) Heatmap of genes regulated by mevastatin in human primary keratinocytes. (B) Gene ontology analysis of biological processes enriched in mevastatin-treated keratinocytes. Diagram of RNA-Seq data showing mevastatin modulation of EGF signaling pathway by inhibiting cell proliferation while inducing cell migration in human keratinocytes (HEKs). Genes in red indicate mevastatin-induced genes involved in migration and genes in green indicate mevastatin-inhibited genes involved in proliferation. (C) Western blot and quantification of pEGFR (Y1173) and total EGFR and downstream effector pERK and total ERK in HEKs treated with 5 μM mevastatin for 48 hours (n = 6). Mevastatin significantly induced p-EGFR and its downstream effector p-ERK. Data are represented as mean ± SD and were analyzed by Student’s t test; *P < 0.05. (D) Confirmation of RNA-Seq data by qPCR of proliferation and migration genes known to be regulated by EGF signaling in HEKs treated with mevastatin (n = 6). Mevastatin inhibited genes involved in cell proliferation and induced genes involved in migration. Data are represented as mean ± SD and were analyzed by Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. (E) Western blot and quantification of mevastatin-induced migratory genes (ArhGEF1, Rac2) and proliferation genes (Cyclin B1) suppressed by mevastatin. (F) HEK scratch assay and cell proliferation assay treated in the presence or absence of 25 ng/mL EGF for 24 hours. 50 nM of PD 0332991, a CDK4 inhibitor, served as a control for cell proliferation assay. Mevastatin stimulated keratinocyte migration while inhibiting cell proliferation even in the presence of EGF. Data are represented as mean ± SD and were analyzed by a 1-way ANOVA followed by Holm-Sidak’s post hoc test, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: For immunofluorescence staining, tissue sections of discarded DFUs were treated with mevastatin as described above and used for staining with anti–phospho-EGFR (Y1173) (53A5) (rabbit, 1:100; Cell Signaling Technology; 4407) or anti-Cav1 (D46G3) (rabbit, 1:200; Cell Signaling Technology; 3267). pEGFR (Y1173) and Cav1 staining was visualized with Alexa Fluor 488–conjugated goat anti–rabbit antibody (1:500; Invitrogen; A11008) and mounted with Prolong DAPI Gold antifade reagent (Invitrogen) to visualize cell nuclei.

Techniques: RNA Sequencing Assay, Migration, Western Blot, Wound Healing Assay, Proliferation Assay

Journal: JCI Insight

Article Title: Mevastatin promotes healing by targeting caveolin-1 to restore EGFR signaling

doi: 10.1172/jci.insight.129320

Figure Lengend Snippet: (A and B) Western blot and quantification of pEGFR (Y1173) and total EGFR in acute healthy wounds (AWs) (n = 5) and diabetic foot ulcers (DFUs) (n = 6). pEGFR is downregulated in DFUs compared with AWs. Data are represented as mean ± SEM and were analyzed by Student’s t test; *P < 0.05. (C and D) Western blot and quantification of p-EGFR and total EGFR from samples obtained from the nonhealing edge of patients with DFUs treated with 5 μM mevastatin for 48 hours (n = 5). Mevastatin significantly induced pEGFR in samples obtained from the nonhealing edge of DFUs compared with vehicle-treated control. Data are represented as mean ± SEM and were analyzed by a ratio-paired t test; **P < 0.01. (E) Immunofluorescence staining of pEGFR in mevastatin-treated DFUs. Mevastatin strongly induced p-EGFR compared with vehicle-treated control. Scale bar: 100 μm.

Article Snippet: For immunofluorescence staining, tissue sections of discarded DFUs were treated with mevastatin as described above and used for staining with anti–phospho-EGFR (Y1173) (53A5) (rabbit, 1:100; Cell Signaling Technology; 4407) or anti-Cav1 (D46G3) (rabbit, 1:200; Cell Signaling Technology; 3267). pEGFR (Y1173) and Cav1 staining was visualized with Alexa Fluor 488–conjugated goat anti–rabbit antibody (1:500; Invitrogen; A11008) and mounted with Prolong DAPI Gold antifade reagent (Invitrogen) to visualize cell nuclei.

Techniques: Western Blot, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Analysis of Epithelial Growth Factor-Receptor (EGFR) Phosphorylation in Uterine Smooth Muscle Tumors: Correlation to Mucin-1 and Galectin-3 Expression

doi: 10.3390/ijms14034783

Figure Lengend Snippet: Salient features of the antibodies used in the current study.

Article Snippet: pEGFR-Y1173 , polyclonal , Rabbit IgG , 1.0 mg/mL , R & D Systems, USA.

Techniques: Concentration Assay