Construction of pCDH-tdTomato Expression Plasmid

To construct pCDH-tdTomato expression plasmid, full-length tdTomato was amplified from the pCDH-EF1-Luc2-P2A-tdTomato (Addgene, 72486) with a pair of primers (Forward: 5'-GCTAGCCCAATCATTTAAATATAACTT-3', Reverse: 5'-GCGGCCGCTTACTTGTACAGCTCGTCCATGC-3'), and then cloned into the pCDH-CMV-MCS-EF1α-Neo vector at the Nhe1 and Not1 sites. The sequence of inserted tdTomato was confirmed by DNA sequencing. The constitutively active STAT3 (STAT3-C-Flag) and pCDH-luciferase constructs were generated as previously described^{7 (link),56 (link)}. Specific shRNAs against BACE1 (shBACE1; #1: TRCN0000000277, #2: TRCN0000000279) or a non-targeting shRNA (shNT; SHC002) were purchased from Sigma-Aldrich. The lentivirus packaging vectors (ps-PAX2, 12260; and pCI-VSVG, 1733) were from Addgene. The plasmids for expressing human BACE1 with the C-terminal His-tag (HG10064-CH) and human IL-6R with the C-terminal Flag-tag (HG10398-CF) were obtained from Sino Biological.

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Zhai K., Huang Z., Huang Q., Tao W., Fang X., Zhang A., Li X., Stark G.R., Hamilton T.A, & Bao S. (2021). Pharmacological Inhibition of BACE1 Suppresses Glioblastoma Growth by Stimulating Macrophage Phagocytosis of Tumor Cells. Nature cancer, 2(11), 1136-1151.

Publication 2021

pCDH-EF1-Luc2-P2A-tdTomato pCDH-CMV-MCS-EF1α-Neo vector shBACE1; #1: TRCN0000000277 TRCN0000000279 shNT; SHC002 ps-PAX2, 12260 HG10064-CH HG10398-CF

Bace1 Biological Human Il 6r Lentivirus Luciferase Nhe1 Pax2 Plasmids Primers Shrna Sino Stat3 Tdtomato Vectors

Corresponding Organization :

Other organizations : Cleveland Clinic Lerner College of Medicine, Case Western Reserve University

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Variable analysis

independent variables

Overexpression of STAT3-C-Flag
Knockdown of BACE1 using shBACE1 #1 and #2

dependent variables

Expression of tdTomato

control variables

Use of the pCDH-CMV-MCS-EF1α-Neo vector
Use of the non-targeting shRNA (shNT) as a control for shBACE1
Use of the pCDH-luciferase construct as a previously described control

controls

Positive control: pCDH-luciferase construct
Negative control: non-targeting shRNA (shNT)

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