n terminal gst tag  (Sino Biological)

Journal: Nucleic Acids Research

Article Title: EGFR-mediated HSP70 phosphorylation facilitates PCNA association with chromatin and DNA replication

doi: 10.1093/nar/gkae938

Figure Lengend Snippet: EGFR interacts and phosphorylates HSP70. ( A ) and ( B ) HSP70–EGFR interaction in HCC827 cells. The HSP70–EGFR complex was immunoprecipitated from HCC827 cell lysates using antibodies against HSP70 (panel A) or EGFR (panel B), respectively. The immunoprecipitated HSP70–EGFR complex was analyzed by western blot. ( C ) In vitro EGFR kinase assay detecting Y41 phosphorylation of HSP70. Purified EGFR kinase domain protein was used as the enzyme and purified Flag-tagged HSP70 (WT or Y41F) was used as the protein substrate. HSP70 Y41 phosphorylation was evaluated by western blot using the antibody against Y41 phosphorylated HSP70. ( D ) Enhancement of HSP70 phosphorylation by EGF. HCC827 cells expressing Flag-tagged WT or Y41F HSP70 were untreated or treated with EGF (100 ng/ml, 1 h). The phosphorylation of Flag-tagged HSP70 (WT or Y41F) was analyzed by Co-IP using anti-Flag-tag antibody and western blot using the antibody against Y41 phosphorylated HSP70. ( E ) Inhibition of HSP70 phosphorylation by EGFR–TKIs. HCC827 cells were treated with EGFR–TKIs osimertinib, erlotinib and gefitinib. The status of EGFR phosphorylation, EGFR level, HSP70 phosphorylation and HSP70 level were analyzed by western blot. GADPH was used as the loading control for western blot.

Article Snippet: Active EGFR kinase domain ( N -terminal GST-tagged human EGFR kinase domain, amino acids 696-end) was purchased from Millipore Sigma (Catalog No.: 14–531).

Techniques: Immunoprecipitation, Western Blot, In Vitro, Kinase Assay, Phospho-proteomics, Purification, Expressing, Co-Immunoprecipitation Assay, FLAG-tag, Inhibition, Control