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. 2016 Jan 29;48(1):e206. doi: 10.1038/emm.2015.102

Figure 1.

Figure 1

CHMP5 knockdown enhances TCR-induced signaling, activating NF-κB. (a) Control (Ctrl) and CHMP5KD Jurkat cells generated by control shRNA lentiviral particles and CHMP5 shRNA lentiviral particles, respectively, were stimulated with anti-CD3/anti-CD28 for different times, as described in Materials and methods section. Cell extracts were analyzed by western blotting for CHMP5, pho-Lck, Lck, pho-ZAP-70, ZAP-70, pho-PKCθ, PKCθ, pho-IKKαβ, IKKα and GAPDH as a loading control. (b, c) Relative pho-PKCθ (b) and pho-IKKαβ (c) levels were evaluated by Image J (lower panels). All error bars represent ±s.d. of the mean from triplicate samples. (d) Control (Ctrl) and CHMP5KD Jurkat cells were transfected with pBIIx-luc NF-κB-dependent reporter together with the Renilla luciferase vector. After 36 h, cells were stimulated with anti-CD3/anti-CD28 at different times and a luciferase assay was performed. All error bars represent ±s.d. of the mean from triplicate samples. *P<0.01. (e, f) Ctrl and CHMP5KD Jurkat cells were stimulated with anti-CD3/anti-CD28 for different times or PMA as a positive control. Nuclear extracts were prepared and analyzed for p65 (e) and p50 (f) binding to an NF-κB consensus oligonucleotide. All error bars represent ±s.d. of the mean from triplicate samples. *P<0.01 and **P<0.05.