HEK293T, A172, U87 and SKNSH cells were maintained in Eagle’s MEM (U87, SKNSH), DMEM (A172, HEK293T), or L-15 (SW620) media (Invitrogen), all with 10% Fetal Bovine Serum (Hyclone). Cells were transfected with PEI or Turbofect (Fisher) according to the manufacturer’s instructions. Protein expression was analyzed by western blot with antibodies to the Flag (Sigma, Flag M2) or V5 epitope tag (Cell Signaling, 13202). Expression of YAP1 was monitored using a YAP/TAZ specific antibody from Santa Cruz (SC-101119), and HSP90 was detected with an antibody from Cell Signaling (#4874). U87 and A172 cells were transduced with lentiviral vectors under standard conditions using polybrene, and 72 hours after infection cells were subjected to selection with 1μg/ml Puromycin or 2μg/ml Blasticidin, or 0.5μg/ml Puromycin plus 2μg/ml Blasticidin. Assays on infected cells were performed after at least 10 days in selection. A172 and U87 cells were authenticated by STR profiling, in accordance with ICLAC guidelines, at the University of Arizona Genetics Core.
V5 epitope tag
Manufactured by Cell Signaling Technology
The V5 epitope tag is a short peptide sequence that can be fused to recombinant proteins to facilitate their detection and purification. It provides a standardized approach for tagging proteins of interest.
Automatically generated - may contain errors
Lab products found in correlation
Turbofect, by Thermo Fisher Scientific (1 mentions)
Flag m2, by Cell Signaling Technology (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Streptavidin hrp conjugate, by Thermo Fisher Scientific (1 mentions)
Serca2a, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
2 protocols using v5 epitope tag
1
Cell Line Maintenance and Transduction
2
Western Blot Analysis of Ion Channels
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Forty micrograms of total protein lysates were resolved by SDS-PAGE, transferred onto nitrocellulose membranes and blocked with BSA. The membranes were subsequently incubated overnight at 4°C in blocking buffer with the corresponding primary antibody: Cavβ2b (1:250, homemade), Cavβ2 (1:1,000, Novus Biologicals, NBP1 86680), Cav1.2 (1:500, Alomone Labs, ACC-003), RyR2 (1:1,000, Thermo Fisher Scientific, MA3-916), V5 epitope tag (1:1,000, Cell Signaling, #13203), SERCA2a (1:1,000, Thermo Fisher Scientific, MA3-919) and GAPDH (1:5,000, Cell Signaling, #2118). Afterwards, the membranes were washed with TBS-Tween and incubated with anti-rabbit IgG or anti-mouse IgG secondary antibodies conjugated to horseradish peroxidase (HRP) for 1 h at RT. For the biotinylated proteins, the membranes were incubated with streptavidin-HRP conjugate (1: 2,000, Thermo Fisher Scientific) during 2 h at RT. After washing, all the membranes were developed using the PierceTM ECL Western Blotting Substrate (Thermo Fisher Scientific).
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