Skip to main content
. 2023 Jan 27;13:1079962. doi: 10.3389/fimmu.2022.1079962

Figure 5.

Figure 5

SIRT2-PFKP in vivo and in vitro interaction. (A) RAW264.7 cell macrophages (RAW) ± LPS. IP of whole-cell lysates using an anti-SIRT2 antibody followed by IB analysis of PFKP and SIRT2. IP with isotype IgG control antibody was used as a negative control. (B) Western blot analysis of PFKP and SIRT2 in the whole cell lysate used as input for the SIRT2 IP. (C) In-vitro interaction between SIRT2 and PFKP using SPR. SIRT2 protein immobilized onto sensor chip and the PFKP was flowed at various concentration (15.62, 31.25, 62.5, 125, 250, 500 and 1000nM). The response units on Y axis (RU) represent quantitative assessment of protein-protein interaction. (D) wtPFKP and control plasmid transfection and IP, using turbo-GFP-trap in HEK293T cells, in presence or absence of SIRT2 followed by IB analysis of acetyl lysine, turbo-GFP-wtPFKP and control for PFKP (turbo-GFP). HEK293T cell lysate without transfection used as a negative control. (E) Western blot analysis of control-PFKP (Turbo-GFP), wtPFKP (Turbo-GFP-wtPFKP), DDK-SIRT2 and CPA in whole cell lysate used as input for the turbo-GFP IP. (F) wtPFKP transfection and IP using magnetic-TUBEs in HEK293T cells, in presence or absence of SIRT2 followed by IB analysis of ubiquitination. (G) Western blot analysis of turbo-GFP wtPFKP, DDK-SIRT2, turbo-GFP and CPA in whole cell lysate used as an input for the TUBE IP.