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