Selected article for: "gfp control and sample buffer"
Author: Lin, Mingqun; Liu, Hongyan; Xiong, Qingming; Niu, Hua; Cheng, Zhihui; Yamamoto, Akitsugu; Rikihisa, Yasuko
Title: Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase
  • Document date: 2016_8_19
    • ID: x5y551c8_59
    Snippet: Etf-1-GFP or GFP control was transfected with Fugene HD into HEK293 or RF/6A cells during exponential growth. At 8 h p.t., E. chaffeensis or A. phagocytophilum, which were freshly isolated as described, 43 were added to infect transfected cells. 37, 43 Bacteria that were not internalized were removed at 1 d p.i., and cells were harvested at 2 d p.i. Bacterial numbers in each sample were determined by qPCR using specific primers for E. chaffeensis.....
    Document: Etf-1-GFP or GFP control was transfected with Fugene HD into HEK293 or RF/6A cells during exponential growth. At 8 h p.t., E. chaffeensis or A. phagocytophilum, which were freshly isolated as described, 43 were added to infect transfected cells. 37, 43 Bacteria that were not internalized were removed at 1 d p.i., and cells were harvested at 2 d p.i. Bacterial numbers in each sample were determined by qPCR using specific primers for E. chaffeensis 16S rDNA and normalized against the level of expression of human GAPDH. 127 Samples were subjected to western blotting using antibodies against A. phagocytophilum P44 and ACT/actin. HEK293 cells cultured in a 12-well plate were transfected with plasmids encoding pEGFP, pEGFP-RAB5 (WT, DN, or CA), pcDNA3.1-MYC-SGSM3 (RABGAP5), 105 or pcDNA3.1-MYC-SGSM3 R165A , 105 and infected with E. chaffeensis at 1 d p. t. for 2 d. Cells were lysed in 1£ SDS sample buffer, sonicated, and examined by western blotting using antibodies against EGFP (for RAB5), MYC/c-Myc (for SGSM3), and E. chaffeensis P28.

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