chx 94271  (Amresco)

Journal: EBioMedicine

Article Title: Systematic identification of CDC34 that functions to stabilize EGFR and promote lung carcinogenesis

doi: 10.1016/j.ebiom.2020.102689

Figure Lengend Snippet: CDC34 inhibits the ubiquitination of EGFR and protects it from proteolytic degradation. (a) qRT-PCR analysis of CDC34 and EGFR in A549 and H1975 cells 72 h after si CDC34 transfection. Error bars, sd; P values, Student's t -test. (b) Cycloheximide (CHX) chase assay to measure EGFR half-life in H460 (in the absence and presence of EGF) and H1975 cells transfected with si CDC34- 1# (lower panel). The relative expression values of EGFR were the results determined by densitometry analysis normalized to Actin. Error bars, sd. (c) HCC827 cells were transfected with 50 nM si CDC34- 1# for 24 h, treated with erlotinib for 24 h, and lyszed for Western blot assays using indicated antibodies. (d) Western blot analysis of the EGFR expression in H460 cells transfected with si CDC34- 1# in the presence or absence of MG132. (e) A549 and H1975 cells were transfected with si CDC34 and HA-Ub, lysed, and the lysates were subjected to immunoprecipitation (IP) and immunoblot using indicated antibodies. (f) A549 cells were transfected with Flag- CDC34 and HA-Ub, and lysed for IP and immunoblot assays. (g) H460 cells were transfected with indicated constructs, treated with MG132, and lysed for IP and immunoblot assays. (h) The cells were transfected with CDC34 and HA-Ub, and lysed for IP and immunoblot assays using indicated antibodies. (i) Western blot analysis of EGFR in #1 si CDC34 -transfected H460 cells in the presence or absence of chloroquine (CQ). (j) HCC827 cells were transfected with siCDC34 and HA-Ub for 24 h, treated with or without chloropuine for additional 24 h, lyzed, and subjected to IP and immunoblot using indicated antibodies. (k) H460, A549, and H1975 cells transfected with si CDC34 were lysed and subjected to Co-IP and immunoblotting using indicated antibodies. (l) H460 cells were transfected with CDC34 (left) or CBL (right) in the absence or presence of EGF for 48 h, and lysed for Co-IP and immunoblot using indicated antibodies. (m) In vitro Co-IP experiments using Flag-EGFR purified from 293T cells, His-CDC34 (from E. coli), and increasing amount of purified His-c-Cbl (from E. coli) proteins and indicated antibodies. (n) In vitro Co-IP experiments using Flag-EGFR purified from 293T cells, His-c-Cbl (from E. coli), and increasing amount of purified His-CDC34 (from E. coli) proteins and indicated antibodies. (o) 293T cells were transfected with the wild type or mutant (Y1045F) Flag- EGFR ICD for 48 h, lyzed, and subjected to IP and immunobloting using indicated antibodies. (p) si CDC34 -mediated loss of EGFR is rescued by knockdown of CBL . A549 cells were transfected with si CDC34 and/or si CBL for 72 h. Before lysis for immunoblot assays, the cells were serum-starved for 3 h and then stimulated by 50 ng/mL EGF for 15 min.

Article Snippet: Reagents used included cycloheximide (CHX) (#94271, Amresco Inc., Solon, OH, USA), erlotinib (#HY-12008, MedChemExpress, USA), epoxomicin (#A2606, APExBIO, USA), Universal Tyrosine Kinase Assay Kit (#MK410, Clontech, Palo Alto, CA), MG132 (Sigma, #SML1135), chloroquine (Sigma, #PHR-1258), BaP (Sigma, #B1760), BAA (Sigma, #B2209), and DBA (Sigma, #91861).

Techniques: Ubiquitin Proteomics, Quantitative RT-PCR, Transfection, Expressing, Western Blot, Immunoprecipitation, Construct, Co-Immunoprecipitation Assay, In Vitro, Purification, Mutagenesis, Knockdown, Lysis